Time-release and micro-dose formulations for topical application of estrogen and estrogen analogs or other estrogen receptor modulators in the treatment of dry eye syndrome, and methods of preparation and application

ABSTRACT

A topical application formulation of estrogen and estrogen analogs or other estrogen receptor modulators is disclosed for the treatment of primary or secondary dry eye syndrome (also known as keratoconjunctivitis sicca (KCS)). Preferred formulations include 17-β-estradiol and its derivatives in lipid, liposomes, polymers, or aqueous or non-aqueous vehicles for the topical treatment of the ocular surface tissues particularly as time-release or micro-dose formulations. These formulations may also be useful in treating other conditions where KCS may occur, such as post-operative refractive surgery and corneal transplant patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase patent application ofPCT/US2010/039250, filed Jun. 18, 2010, which claims the benefit of U.S.Provisional Application No. 61/218,899, filed Jun. 19, 2009, each ofwhich is hereby incorporated by reference, in its entirety.

BACKGROUND OF THE INVENTION

1. Field

This invention relates to the topical application of estrogen andestrogen analogs and other estrogen receptor modulators in the treatmentof primary or secondary dry eye syndrome (also known askeratoconjunctivitis sicca (KCS)) and, in certain embodiments, to thepreparation and application of 17-β-estradiol and its derivatives inlipid, liposomes, polymers, or aqueous or non-aqueous vehicles for thetopical treatment of the ocular surface tissues particularly astime-release or micro-dose formulations. This invention may also beuseful in treating other conditions where KCS may occur, such aspost-operative refractive surgery and corneal transplant patients.

2. Related Art

The high incidence of keratoconjunctivitis sicca in the population ofpostmenopausal women is attended by symptoms ranging from mild foreignbody sensation to frank pain and visual loss due to ocular surfaceabnormalities.

It is known that tear film quality depends on fine regulatory mechanismsaffected by neuronal and hormonal influences. Indeed, receptors forandrogens, estrogens, progesterone and prolactin have been identified inseveral ocular tissues in the rat, rabbit and in humans. These hormonesinfluence the immune system, the morphology and secretory functions oflacrimal glands, the morphology and function of the conjunctiva, and thefunctioning of Meibomian glands. The influence of hormone replacementtherapy in menopausal women remains unclear, as some authors support theidea that hormones improve the quality and the volume of tear film,whereas others have argued that they increase the risk of dry eye.Finally, knowledge of the interactions between the hormones thatinfluence the function of the lacrimal gland is an essential element forthe understanding of the regulation of lacrimal gland function.Additional data suggest that optimal bioavailable androgen levels areessential for normal lacrimal gland function and that prolactin andestrogens also play important roles in providing a hormonal milieu thatcontributes to normal lacrimal gland function (Oprea, L., Tiberghien,A., Creuzot-Garcher, C., Baudouin, C., Hormonal Regulatory Influence inTear Film, J. Fr. Ophtalmol., October 27(8):93341 (2004)).

The standard treatment with artificial lubricants provides temporarysymptomatic relief in most cases, but does not address the cause of thedry eyes. While there has been described treatment of post menopausalfemales with dry eye syndrome using oral Premarin therapy, the oral orparenteral administration of estrogen can frequently produce sideeffects such as vaginal bleeding, breast tenderness and other undesiredeffects and the therapeutic effects derived from oral therapy do notjustify the risk. Further, such oral, dermal (e.g., application to theskin of the outer eyelid) or parenteral administration implicates theentire body structure in an indeterminate effort to secure an effect ina localized area (the eye), in the absence of any data relating thelevel of estrogen introduced into the blood stream to the level, if any,resulting in the tear fluid (it is known generally, that estrogenconcentrations in the eye to be in the range of about 10% of serumlevels). Conservative medicine would indicate the desirability oflimiting the specific effect of the hormone to the target site ifpossible.

As the data in U.S. Pat. No. 6,096,733 demonstrates, the effectiveconcentration of 17-β-estradiol in solution can be as low as 0.1% andcontinue to be effective regardless of the presence or absence ofconcomitant oral estrogen therapy in post-menopausal women. This lowerdose range is especially useful in providing eye drops that will containa concentration of 17-β-estradiol that is low enough to be both safe andeffective (the medical aspect) yet has the potential to be approved bythe FDA for use in non-prescription (OTC) based formulations (thecommercial aspect). However, the low dose of 0.1% was applied three orfour times per day in U.S. Pat. No. 6,096,733. Thus, U.S. Pat. No.6,096,733, while providing a low dose formulation, did not provideexamples of a time release or micro-dose formulation that could be usedonly at lower frequency.

In addition, the low dose formulation in the U.S. Pat. No. 6,096,733exhibits side effect due to systemic effects of the 17-β-estradioladministered topically. When a lower does (0.05% 17-β-estradiol) wastested in a Phase IIb trial, it showed lower efficacy than the 0.1% dosefor all but one of the signs and symptoms recorded for the trial. Thus,there is a need for formulations that can provide an efficacious dosewithout the systemic side effects, preferably with a lower frequency ofdosing.

BRIEF SUMMARY OF THE INVENTION

Accordingly, it is a principal object of this invention to providetreatment by topical application of 17-β-estradiol suspended ordissolved or otherwise incorporated in a suitable vehicle formulated forlong-term release and/or micro-dose to the conjunctival surface of theeye, the inner eyelid or the outer eyelid to alleviate dry-eye syndromeor KCS that need be delivered only at twice per day or lower frequencywhile retaining full efficacy. Surprisingly, these timed releaseformulations are efficacious at a much lower dose than the formulationprovided in U.S. Pat. No. 6,096,733. Thus, these novel formulations meetboth needs by providing an efficacious dose without the systemic sideeffects and by providing a sustained release profile that allows a lowerdose frequency.

One aspect of the invention includes a topical applicationpharmaceutical composition for timed release comprising estrogen or anestrogen analog wherein the timed release is sufficient for delivery ofa therapeutically effective concentration for alleviation ofkerato-conjunctivitis sicca (dry eye syndrome) when administered twicedaily or less frequently. In certain embodiments, the pharmaceutical isin the form of a semi-solid insert for the cul-de-sac of the eye, a gel,or a semi-solid ointment. In other embodiments which may be combinedwith the preceding embodiments, the topical application pharmaceuticalincludes a bioadhesive viscoelastic compound. In other embodiments whichmay be combined with the preceding embodiments that include abioadhesive viscoelastic compound, the bioadhesive viscoelastic compoundis in a concentration sufficient to achieve the timed release. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is selected from the group consisting ofpolycarbophil, hyaluronate, chitosan, a polysaccharide andpolycarboxylated polymer, carboxylmethyl cellulose, hydroxypropyl methylcellulose, liposomes, a polysaccharide and a positively-chargedsubmicron emulsion. In other embodiments which may be combined with thepreceding embodiments that include a polysaccharide, the polysaccharideis xanthan gum, which optionally can be between about 0.1% to about 1%(w/w) or about 0.6% (w/w). In other embodiments which may be combinedwith the preceding embodiments that include carboxylmethyl cellulose,the carboxylmethyl cellulose is LACRISERT™. In other embodiments whichmay be combined with the preceding embodiments that include apositively-charged submicron emulsion, the positively-charged submicronemulsion is NOVASORB™ or CATIONORM™. In other embodiments which may becombined with the preceding embodiments that include liposomes, theliposomes are multi-vesicular. In other embodiments which may becombined with the preceding embodiments that include multi-vesicularliposomes, the multi-vesicular liposomes are DEPOFOAM™. In otherembodiments which may be combined with the preceding embodiments thatinclude a polysaccharide and polycarboxylated polymer, thepolysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release is sufficient for delivery of a therapeuticallyeffective concentration for alleviation of kerato-conjunctivitis sicca(dry eye syndrome) when administered once per day or less frequently,every other day or less frequently, every third day or less frequentlyor once per week or less frequently. In other embodiments which may becombined with the preceding embodiments, the timed release is sufficientfor delivery of a therapeutically effective concentration foralleviation of kerato-conjunctivitis sicca (dry eye syndrome) whenadministered once per day, every other day, every third day or once perweek. In other embodiments which may be combined with the precedingembodiments, the timed release comprises a controlled, sustained drugrelease rate which does not vary by more than about 50% for a period ofat least about 6-8 hours after application, at least about 8-12 hoursafter application, at least about 12-24 hours after application, atleast about 24-48 hours after application, or at least about 48-72 hoursafter application. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled,sustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog per hour at least about 6-8hours after application, at least about 8-12 hours after application, atleast about 12-24 hours after application, at least about 24-48 hoursafter application, or at least about 48-72 hours after application. Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich delivers as measured by a Franz diffusion cell model withsimulated tear flow a dose of between 0.1 mg/ml and 1.0 mg/ml ofestrogen or the estrogen analog per hour at least about 6-8 hours afterapplication, at least about 8-12 hours after application, at least about12-24 hours after application, at least about 24-48 hours afterapplication, or at least about 48-72 hours after application. In otherembodiments which may be combined with the preceding embodiments, theestrogen analog is 17-β-estradiol. In other embodiments which may becombined with the preceding embodiments, the therapeutically effectiveamount is between about 0.005% and 5% weight-by-weight of estrogen orthe estrogen analog. In other embodiments which may be combined with thepreceding embodiments, the therapeutically effective amount is betweenabout 0.005% and about 0.1%, 0.005% and less than 0.05%, 0.01% and about0.04%, about 0.05% and about 0.5%, about 0.05% and about 0.1%, about0.1% and about 5%, about 0.5% and 5%, and less than or equal to 0.1%. Inother embodiments which may be combined with the preceding embodiments,the increase in peak blood estradiol concentration after continuous useis less that 30 pg/ml, less than 20 pg/ml, less than 15 pg/ml, less than10 pg/ml or less than 6 pg/ml. In other embodiments which may becombined with the preceding embodiments, the average blood concentrationof estrogen after continuous use is less than 12 m I.U., less than 10 mI.U., less than 8 m I.U., less than 6 m I.U., less than 5 m I.U., lessthan 4 m I.U., or less than 3 m I.U. In other embodiments which may becombined with the preceding embodiments, the topical applicationpharmaceutical further includes a therapeutically effective amount of anandrogen. In other embodiments which may be combined with the precedingembodiments that include an androgen, the androgen is selected from thegroup consisting of testosterone, dihydrotestosterone (also termedallodihydrotestosterone, androstanolone, stanolone, 5alpha-dihydrotestosterone), fluoxymesterone, stanozolol, nortestosteronepropionate, dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Another aspect of the invention includes a topical applicationpharmaceutical composition for timed release comprising17-beta-estradiol or a derivative thereof at a concentration of lessthan 0.05% wherein the timed release is sufficient for delivery of atherapeutically effective concentration for alleviation ofkerato-conjunctivitis sicca (dry eye syndrome) when administered twicedaily or less frequently. In certain embodiments, the pharmaceutical isin the form of a semi-solid insert for the cul-de-sac of the eye, a gel,or a semi-solid ointment. In other embodiments which may be combinedwith the preceding embodiments, the topical application pharmaceuticalincludes a bioadhesive viscoelastic compound. In other embodiments whichmay be combined with the preceding embodiments that include abioadhesive viscoelastic compound, the bioadhesive viscoelastic compoundis in a concentration sufficient to achieve the timed release. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is selected from the group consisting ofpolycarbophil, hyaluronate, chitosan, a polysaccharide andpolycarboxylated polymer, carboxyl methyl cellulose, hydroxypropylmethyl cellulose, liposomes, a polysaccharide and a positively-chargedsubmicron emulsion. In other embodiments which may be combined with thepreceding embodiments that include a polysaccharide, the polysaccharideis xanthan gum, which optionally can be between about 0.1% to about 1%(w/w) or about 0.6% (w/w). In other embodiments which may be combinedwith the preceding embodiments that include carboxylmethyl cellulose,the carboxylmethyl cellulose is LACRISERT™. In other embodiments whichmay be combined with the preceding embodiments that include apositively-charged submicron emulsion, the positively-charged submicronemulsion is NOVASORB™ or CATIONORM™. In other embodiments which may becombined with the preceding embodiments that include liposomes, theliposomes are multi-vesicular. In other embodiments which may becombined with the preceding embodiments that include multi-vesicularliposomes, the multi-vesicular liposomes are DEPOFOAM™. In otherembodiments which may be combined with the preceding embodiments thatinclude a polysaccharide and polycarboxylated polymer, thepolysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release is sufficient for delivery of a therapeuticallyeffective concentration for alleviation of kerato-conjunctivitis sicca(dry eye syndrome) when administered once per day or less frequently,every other day or less frequently, every third day or less frequentlyor once per week or less frequently. In other embodiments which may becombined with the preceding embodiments, the timed release is sufficientfor delivery of a therapeutically effective concentration foralleviation of kerato-conjunctivitis sicca (dry eye syndrome) whenadministered once per day, every other day, every third day or once perweek. In other embodiments which may be combined with the precedingembodiments, the timed release comprises a controlled, sustained drugrelease rate which does not vary by more than about 50% for a period ofat least about 6-8 hours after application, at least about 8-12 hoursafter application, at least about 12-24 hours after application, atleast about 24-48 hours after application, or at least about 48-72 hoursafter application. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled,sustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog per hour at least about 6-8hours after application, at least about 8-12 hours after application, atleast about 12-24 hours after application, at least about 24-48 hoursafter application, or at least about 48-72 hours after application. Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich delivers as measured by a Franz diffusion cell model withsimulated tear flow a dose of between 0.1 mg/ml and 1.0 mg/ml ofestrogen or the estrogen analog per hour at least about 6-8 hours afterapplication, at least about 8-12 hours after application, at least about12-24 hours after application, at least about 24-48 hours afterapplication, or at least about 48-72 hours after application. In otherembodiments which may be combined with the preceding embodiments, theestrogen analog is 17-β-estradiol. In other embodiments which may becombined with the preceding embodiments, the therapeutically effectiveamount is between about 0.005% and less than 0.05% weight-by-weight ofestrogen or the estrogen analog. In other embodiments which may becombined with the preceding embodiments, the therapeutically effectiveamount is between about 0.01% and less than 0.04%, 0.005% and about0.04%, and 0.01% and about 0.04%. In other embodiments which may becombined with the preceding embodiments, the increase in peak bloodestradiol concentration after continuous use is less that 30 pg/ml, lessthan 20 pg/ml, less than 15 pg/ml, less than 10 pg/ml or less than 6pg/ml. In other embodiments which may be combined with the precedingembodiments, the average blood concentration of estrogen aftercontinuous use is less than 12 m I.U., less than 10 m I.U., less than 8m I.U., less than 6 m I.U., less than 5 m I.U., less than 4 m I.U., orless than 3 m I.U. In other embodiments which may be combined with thepreceding embodiments, the topical application pharmaceutical furtherincludes a therapeutically effective amount of an androgen. In otherembodiments which may be combined with the preceding embodiments thatinclude an androgen, the androgen is selected from the group consistingof testosterone, dihydrotestosterone (also termedallodihydrotestosterone, androstanolone, stanolone, 5alpha-dihydrotestosterone), fluoxymesterone, stanozolol, nortestosteronepropionate, dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Yet another aspect of the invention includes methods of treatmentcomprising topical application to an eye of a topical applicationpharmaceutical according to either of the two preceding aspects with anyof their respective combinations of embodiments. In certain embodiments,the kerato-conjunctivitis sicca is associated with post-menopausalsubjects, post-operative refractive surgery patients or cornealtransplant patients.

Still another aspect of the invention includes use of a topicalapplication pharmaceutical according to either of the two precedingtopical application pharmaceutical composition aspects with any of theirrespective combinations of embodiments in the manufacture of amedicament for alleviation of kerato-conjunctivitis sicca (dry eyesyndrome). In certain embodiments, the kerato-conjunctivitis sicca isassociated with post-menopausal subjects, post-operative refractivesurgery patients or corneal transplant patients.

Another aspect of the invention includes methods of alleviation ofkerato-conjunctivitis sicca (dry eye syndrome) comprising administeringby topical application a therapeutically effective dose to an eye of atopical application pharmaceutical, wherein the therapeuticallyeffective dose comprises a volume of less than a standard ophthalmicdrop and wherein the therapeutically effective dose less than 35micrograms of estrogen or an estrogen analog. In certain embodiments,the volume is less than 35 μl, less than 30 μl, less than 25 μl, lessthan 20 μl, less than 15 μl, or between 5 μl and 15 μl. In otherembodiments that may be combined with the preceding embodiments, thedose is less 30 micrograms, less 25 micrograms, less 20 micrograms, less15 micrograms, or less 10 micrograms of estrogen or an estrogen analog.In other embodiments that may be combined with the precedingembodiments, the topical application pharmaceutical is administeredtwice daily or less frequently. In other embodiments that may becombined with the preceding embodiments, the pharmaceutical is in theform of a semi-solid insert for the cul-de-sac of the eye, a gel, or asemi-solid ointment. In other embodiments that may be combined with thepreceding embodiments, the topical application pharmaceutical isadministered once per day or less frequently, every other day or lessfrequently, every third day or less frequently or once per week or lessfrequently. In other embodiments that may be combined with the precedingembodiments, the topical application pharmaceutical is administered onceper day, every other day, every third day or once per week. In otherembodiments that may be combined with the preceding embodiments, theestrogen analog is 17-β-estradiol. In other embodiments which may becombined with the preceding embodiments, the increase in peak bloodestradiol concentration after continuous use is less that 30 pg/ml, lessthan 20 pg/ml, less than 15 pg/ml, less than 10 pg/ml or less than 6pg/ml. In other embodiments that may be combined with the precedingembodiments, the average blood concentration of estrogen aftercontinuous use is less than 12 m I.U., less than 10 m I.U., less than 8m I.U., less than 6 m I.U., less than 5 m I.U., less than 4 m I.U., orless than 3 m I.U. In other embodiments which may be combined with thepreceding embodiments, the method includes a bioadhesive viscoelasticcompound for timed release of the estrogen or estrogen analog. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is in a concentration sufficient to achieve thetimed release. In other embodiments which may be combined with thepreceding embodiments that include a bioadhesive viscoelastic compound,the bioadhesive viscoelastic compound is selected from the groupconsisting of polycarbophil, hyaluronate, chitosan, a polysaccharide andpolycarboxylated polymer, carboxylmethyl cellulose, hydroxypropyl methylcellulose, liposomes, a polysaccharide and a positively-chargedsubmicron emulsion. In other embodiments which may be combined with thepreceding embodiments that include a polysaccharide, the polysaccharideis xanthan gum, which optionally can be between about 0.1% to about 1%(w/w) or about 0.6% (w/w). In other embodiments which may be combinedwith the preceding embodiments that include carboxylmethyl cellulose,the carboxylmethyl cellulose is LACRISERT™. In other embodiments whichmay be combined with the preceding embodiments that include apositively-charged submicron emulsion, the positively-charged submicronemulsion is NOVASORB™ or CATIONORM™. In other embodiments which may becombined with the preceding embodiments that include liposomes, theliposomes are multi-vesicular. In other embodiments which may becombined with the preceding embodiments that include multi-vesicularliposomes, the multi-vesicular liposomes are DEPOFOAM™. In otherembodiments which may be combined with the preceding embodiments thatinclude a polysaccharide and polycarboxylated polymer, thepolysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich does not vary by more than about 50% for a period of at leastabout 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled, thesustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog per hour at least about 6-8hours after application, at least about 8-12 hours after application, atleast about 12-24 hours after application, at least about 24-48 hoursafter application, or at least about 48-72 hours after application. Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich delivers as measured by a Franz diffusion cell model withsimulated tear flow a dose of between 0.1 mg/ml and 1.0 mg/ml ofestrogen or the estrogen analog per hour at least about 6-8 hours afterapplication, at least about 8-12 hours after application, at least about12-24 hours after application, at least about 24-48 hours afterapplication, or at least about 48-72 hours after application. In otherembodiments which may be combined with the preceding embodiments, themethod further includes a therapeutically effective amount of anandrogen. In other embodiments which may be combined with the precedingembodiments that include an androgen, the androgen is selected from thegroup consisting of testosterone, dihydrotestosterone (also termedallodihydrotestosterone, androstanolone, stanolone, 5alpha-dihydrotestosterone), fluoxymesterone, stanozolol, nortestosteronepropionate, dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Still another aspect includes methods of alleviation ofkerato-conjunctivitis sicca (dry eye syndrome) comprising administeringby topical application a therapeutically effective dose to an eye of atopical application pharmaceutical, wherein the therapeuticallyeffective dose comprises a volume of less than 35 μl and wherein thetherapeutically effective dose less than 35 micrograms of17-beta-estradiol or a derivative. In certain embodiments, the volume isless than 30 μl, less than 25 μl, less than 20 μl, less than 15 μl, orbetween 5 μl and 15 μl. In other embodiments that may be combined withthe preceding embodiments, the dose is less 30 micrograms, less 25micrograms, less 20 micrograms, less 15 micrograms, or less 10micrograms of estrogen or an estrogen analog. In other embodiments thatmay be combined with the preceding embodiments, the topical applicationpharmaceutical is administered twice daily or less frequently. In otherembodiments that may be combined with the preceding embodiments, thepharmaceutical is in the form of a semi-solid insert for the cul-de-sacof the eye, a gel, or a semi-solid ointment. In other embodiments thatmay be combined with the preceding embodiments, the topical applicationpharmaceutical is administered once per day or less frequently, everyother day or less frequently, every third day or less frequently or onceper week or less frequently. In other embodiments that may be combinedwith the preceding embodiments, the topical application pharmaceuticalis administered once per day, every other day, every third day or onceper week. In other embodiments which may be combined with the precedingembodiments, the increase in peak blood estradiol concentration aftercontinuous use is less that 30 pg/ml, less than 20 pg/ml, less than 15pg/ml, less than 10 pg/ml or less than 6 pg/ml. In other embodimentsthat may be combined with the preceding embodiments, the average bloodconcentration of estrogen after continuous use is less than 12 m I.U.,less than 10 m I.U., less than 8 m I.U., less than 6 m I.U., less than 5m I.U., less than 4 m I.U., or less than 3 m I.U. In other embodimentswhich may be combined with the preceding embodiments, the methodincludes a bioadhesive viscoelastic compound for timed release of theestrogen or estrogen analog. In other embodiments which may be combinedwith the preceding embodiments that include a bioadhesive viscoelasticcompound, the bioadhesive viscoelastic compound is in a concentrationsufficient to achieve the timed release. In other embodiments which maybe combined with the preceding embodiments that include a bioadhesiveviscoelastic compound, the bioadhesive viscoelastic compound is selectedfrom the group consisting of polycarbophil, hyaluronate, chitosan, apolysaccharide and polycarboxylated polymer, carboxylmethyl cellulose,hydroxypropyl methyl cellulose, liposomes, a polysaccharide and apositively-charged submicron emulsion. In other embodiments which may becombined with the preceding embodiments that include a polysaccharide,the polysaccharide is xanthan gum, which optionally can be between about0.1% to about 1% (w/w) or about 0.6% (w/w). In other embodiments whichmay be combined with the preceding embodiments that includecarboxylmethyl cellulose, the carboxyl methyl cellulose is LACRISERT™.In other embodiments which may be combined with the precedingembodiments that include a positively-charged submicron emulsion, thepositively-charged submicron emulsion is NOVASORB™ or CATIONORM™. Inother embodiments which may be combined with the preceding embodimentsthat include liposomes, the liposomes are multi-vesicular. In otherembodiments which may be combined with the preceding embodiments thatinclude multi-vesicular liposomes, the multi-vesicular liposomes areDEPOFOAM™. In other embodiments which may be combined with the precedingembodiments that include a polysaccharide and polycarboxylated polymer,the polysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich does not vary by more than about 50% for a period of at leastabout 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled,sustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog per hour at least about 6-8hours after application, at least about 8-12 hours after application, atleast about 12-24 hours after application, at least about 24-48 hoursafter application, or at least about 48-72 hours after application. Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich delivers as measured by a Franz diffusion cell model withsimulated tear flow a dose of between 0.1 mg/ml and 1.0 mg/ml ofestrogen or the estrogen analog per hour at least about 6-8 hours afterapplication, at least about 8-12 hours after application, at least about12-24 hours after application, at least about 24-48 hours afterapplication, or at least about 48-72 hours after application. In otherembodiments which may be combined with the preceding embodiments, themethod further includes a therapeutically effective amount of anandrogen. In other embodiments which may be combined with the precedingembodiments that include an androgen, the androgen is selected from thegroup consisting of testosterone, dihydrotestosterone (also termedallodihydrotestosterone, androstanolone, stanolone, 5alpha-dihydrotestosterone), fluoxymesterone, stanozolol, nortestosteronepropionate, dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Yet another aspect includes use of a topical application pharmaceuticalfor use in either of the two preceding aspects with any of theirrespective combinations of embodiments in the manufacture of amedicament for alleviation of kerato-conjunctivitis sicca (dry eyesyndrome). In certain embodiments, the kerato-conjunctivitis sicca isassociated with post-menopausal subjects, post-operative refractivesurgery patients or corneal transplant patients.

Another aspect of the invention includes a topical applicationpharmaceutical composition for timed release comprising estrogen or anestrogen analog or other estrogen receptor modulator wherein the timedrelease is sufficient for delivery of a therapeutically effectiveconcentration for alleviation of kerato-conjunctivitis sicca (dry eyesyndrome) when administered twice daily or less frequently. In certainembodiments, the pharmaceutical is in the form of a semi-solid insertfor the cul-de-sac of the eye, a gel, or a semi-solid ointment. In otherembodiments which may be combined with the preceding embodiments, thetopical application pharmaceutical includes a bioadhesive viscoelasticcompound. In other embodiments which may be combined with the precedingembodiments that include a bioadhesive viscoelastic compound, thebioadhesive viscoelastic compound is in a concentration sufficient toachieve the timed release. In other embodiments which may be combinedwith the preceding embodiments that include a bioadhesive viscoelasticcompound, the bioadhesive viscoelastic compound is selected from thegroup consisting of polycarbophil, hyaluronate, chitosan, apolysaccharide and polycarboxylated polymer, carboxyl methyl cellulose,hydroxypropyl methyl cellulose, liposomes, a polysaccharide and apositively-charged submicron emulsion. In other embodiments which may becombined with the preceding embodiments that include a polysaccharide,the polysaccharide is xanthan gum, which optionally can be between about0.1% to about 1% (w/w) or about 0.6% (w/w). In other embodiments whichmay be combined with the preceding embodiments that includecarboxylmethyl cellulose, the carboxylmethyl cellulose is LACRISERT™. Inother embodiments which may be combined with the preceding embodimentsthat include a positively-charged submicron emulsion, thepositively-charged submicron emulsion is NOVASORB™ or CATIONORM™. Inother embodiments which may be combined with the preceding embodimentsthat include liposomes, the liposomes are multi-vesicular. In otherembodiments which may be combined with the preceding embodiments thatinclude multi-vesicular liposomes, the multi-vesicular liposomes areDEPOFOAM™. In other embodiments which may be combined with the precedingembodiments that include a polysaccharide and polycarboxylated polymer,the polysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release is sufficient for delivery of a therapeuticallyeffective concentration for alleviation of kerato-conjunctivitis sicca(dry eye syndrome) when administered once per day or less frequently,every other day or less frequently, every third day or less frequentlyor once per week or less frequently. In other embodiments which may becombined with the preceding embodiments, the timed release is sufficientfor delivery of a therapeutically effective concentration foralleviation of kerato-conjunctivitis sicca (dry eye syndrome) whenadministered once per day, every other day, every third day or once perweek. In other embodiments which may be combined with the precedingembodiments, the timed release comprises a controlled, sustained drugrelease rate which does not vary by more than about 50% for a period ofat least about 6-8 hours after application, at least about 8-12 hoursafter application, at least about 12-24 hours after application, atleast about 24-48 hours after application, or at least about 48-72 hoursafter application. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled,sustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog or other estrogen receptormodulator per hour at least about 6-8 hours after application, at leastabout 8-12 hours after application, at least about 12-24 hours afterapplication, at least about 24-48 hours after application, or at leastabout 48-72 hours after application. In other embodiments which may becombined with the preceding embodiments, the timed release comprises acontrolled, sustained drug release rate which delivers as measured by aFranz diffusion cell model with simulated tear flow a dose of between0.1 mg/ml and 1.0 mg/ml of estrogen or the estrogen analog per hour atleast about 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the estrogen analog is 17-β-estradiol. In otherembodiments which may be combined with the preceding embodiments, thetherapeutically effective amount is between about 0.005% and 5%weight-by-weight of estrogen or the estrogen analog or other estrogenreceptor modulator. In other embodiments which may be combined with thepreceding embodiments, the therapeutically effective amount is betweenabout 0.005% and about 0.1%, 0.005% and less than 0.05%, 0.01% and about0.04%, about 0.05% and about 0.5%, about 0.05% and about 0.1%, about0.1% and about 5%, about 0.5% and 5%, and less than or equal to 0.1%. Inother embodiments which may be combined with the preceding embodiments,the increase in peak blood estradiol concentration after continuous useis less that 30 pg/ml, less than 20 pg/ml, less than 15 pg/ml, less than10 pg/ml or less than 6 pg/ml. In other embodiments which may becombined with the preceding embodiments, the average blood concentrationof estrogen after continuous use is less than 12 m I.U., less than 10 mI.U., less than 8 m I.U., less than 6 m I.U., less than 5 m I.U., lessthan 4 m I.U., or less than 3 m I.U. In other embodiments which may becombined with the preceding embodiments, the topical applicationpharmaceutical further includes a therapeutically effective amount of anandrogen. In other embodiments which may be combined with the precedingembodiments that include an androgen, the androgen is selected from thegroup consisting of testosterone, dihydrotestosterone (also termedallodihydrotestosterone, androstanolone, stanolone, 5alpha-dihydrotestosterone), fluoxymesterone, stanozolol, nortestosteronepropionate, dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Yet another aspect of the invention includes methods of treatmentcomprising topical application to an eye of a topical applicationpharmaceutical according to the preceding aspect with any of itsrespective combinations of embodiments. In certain embodiments, thekerato-conjunctivitis sicca is associated with post-menopausal subjects,subjects with premature ovarian failure, post-operative refractivesurgery patients, corneal transplant patients or patients with otherconditions that cause dry eye symptoms.

Still another aspect of the invention includes use of a topicalapplication pharmaceutical according to the two preceding topicalapplication pharmaceutical composition aspect with any of its respectivecombinations of embodiments in the manufacture of a medicament foralleviation of kerato-conjunctivitis sicca (dry eye syndrome). Incertain embodiments, the kerato-conjunctivitis sicca is associated withpost-menopausal subjects, premature ovarian failure, post-operativerefractive surgery patients, corneal transplant patients or patientswith other conditions that cause dry eye symptoms.

Another aspect of the invention includes methods of alleviation ofkerato-conjunctivitis sicca (dry eye syndrome) comprising administeringby topical application a therapeutically effective dose to an eye of atopical application pharmaceutical, wherein the therapeuticallyeffective dose comprises a volume of less than a standard ophthalmicdrop and wherein the therapeutically effective dose less than 35micrograms of estrogen or an estrogen analog or other estrogen receptormodulator. In certain embodiments, the volume is less than 35 μl, lessthan 30 μl, less than 25 μl, less than 20 μl, less than 15 μl, orbetween 5 μl and 15 μl. In other embodiments that may be combined withthe preceding embodiments, the dose is less 30 micrograms, less 25micrograms, less 20 micrograms, less 15 micrograms, or less 10micrograms of estrogen or an estrogen analog or other estrogen receptormodulator. In other embodiments that may be combined with the precedingembodiments, the topical application pharmaceutical is administeredtwice daily or less frequently. In other embodiments that may becombined with the preceding embodiments, the pharmaceutical is in theform of a semi-solid insert for the cul-de-sac of the eye, a gel, or asemi-solid ointment. In other embodiments that may be combined with thepreceding embodiments, the topical application pharmaceutical isadministered once per day or less frequently, every other day or lessfrequently, every third day or less frequently or once per week or lessfrequently. In other embodiments that may be combined with the precedingembodiments, the topical application pharmaceutical is administered onceper day, every other day, every third day or once per week. In otherembodiments that may be combined with the preceding embodiments, theestrogen analog is 17-β-estradiol. In other embodiments which may becombined with the preceding embodiments, the increase in peak bloodestradiol concentration after continuous use is less that 30 pg/ml, lessthan 20 pg/ml, less than 15 pg/ml, less than 10 pg/ml or less than 6pg/ml. In other embodiments that may be combined with the precedingembodiments, the average blood concentration of estrogen aftercontinuous use is less than 12 m I.U., less than 10 m I.U., less than 8m I.U., less than 6 m I.U., less than 5 m I.U., less than 4 m I.U., orless than 3 m I.U. In other embodiments which may be combined with thepreceding embodiments, the method includes a bioadhesive viscoelasticcompound for timed release of the estrogen or estrogen analog or otherestrogen receptor modulator. In other embodiments which may be combinedwith the preceding embodiments that include a bioadhesive viscoelasticcompound, the bioadhesive viscoelastic compound is in a concentrationsufficient to achieve the timed release. In other embodiments which maybe combined with the preceding embodiments that include a bioadhesiveviscoelastic compound, the bioadhesive viscoelastic compound is selectedfrom the group consisting of polycarbophil, hyaluronate, chitosan, apolysaccharide and polycarboxylated polymer, carboxylmethyl cellulose,hydroxypropyl methyl cellulose, liposomes, a polysaccharide and apositively-charged submicron emulsion. In other embodiments which may becombined with the preceding embodiments that include a polysaccharide,the polysaccharide is xanthan gum, which optionally can be between about0.1% to about 1% (w/w) or about 0.6% (w/w). In other embodiments whichmay be combined with the preceding embodiments that includecarboxylmethyl cellulose, the carboxyl methyl cellulose is LACRISERT™.In other embodiments which may be combined with the precedingembodiments that include a positively-charged submicron emulsion, thepositively-charged submicron emulsion is NOVASORB™ or CATIONORM™. Inother embodiments which may be combined with the preceding embodimentsthat include liposomes, the liposomes are multi-vesicular. In otherembodiments which may be combined with the preceding embodiments thatinclude multi-vesicular liposomes, the multi-vesicular liposomes areDEPOFOAM™. In other embodiments which may be combined with the precedingembodiments that include a polysaccharide and polycarboxylated polymer,the polysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich does not vary by more than about 50% for a period of at leastabout 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled, thesustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog or other estrogen receptormodulator per hour at least about 6-8 hours after application, at leastabout 8-12 hours after application, at least about 12-24 hours afterapplication, at least about 24-48 hours after application, or at leastabout 48-72 hours after application. In other embodiments which may becombined with the preceding embodiments, the timed release comprises acontrolled, sustained drug release rate which delivers as measured by aFranz diffusion cell model with simulated tear flow a dose of between0.1 mg/ml and 1.0 mg/ml of estrogen or the estrogen analog per hour atleast about 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the method further includes a therapeuticallyeffective amount of an androgen. In other embodiments which may becombined with the preceding embodiments that include an androgen, theandrogen is selected from the group consisting of testosterone,dihydrotestosterone (also termed allodihydrotestosterone,androstanolone, stanolone, 5 alpha-dihydrotestosterone),fluoxymesterone, stanozolol, nortestosterone propionate,dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Still another aspect includes methods of alleviation ofkerato-conjunctivitis sicca (dry eye syndrome) comprising administeringby topical application a therapeutically effective dose to an eye of atopical application pharmaceutical, wherein the therapeuticallyeffective dose comprises a volume of less than 35 μl and wherein thetherapeutically effective dose less than 35 micrograms of17-beta-estradiol or a derivative. In certain embodiments, the volume isless than 30 μl, less than 25 μl, less than 20 μl, less than 15 μl, orbetween 5 μl and 15 μl. In other embodiments that may be combined withthe preceding embodiments, the dose is less 30 micrograms, less 25micrograms, less 20 micrograms, less 15 micrograms, or less 10micrograms of estrogen or an estrogen analog or other estrogen receptormodulator. In other embodiments that may be combined with the precedingembodiments, the topical application pharmaceutical is administeredtwice daily or less frequently. In other embodiments that may becombined with the preceding embodiments, the pharmaceutical is in theform of a semi-solid insert for the cul-de-sac of the eye, a gel, or asemi-solid ointment. In other embodiments that may be combined with thepreceding embodiments, the topical application pharmaceutical isadministered once per day or less frequently, every other day or lessfrequently, every third day or less frequently or once per week or lessfrequently. In other embodiments that may be combined with the precedingembodiments, the topical application pharmaceutical is administered onceper day, every other day, every third day or once per week. In otherembodiments which may be combined with the preceding embodiments, theincrease in peak blood estradiol concentration after continuous use isless that 30 pg/ml, less than 20 pg/ml, less than 15 pg/ml, less than 10pg/ml or less than 6 pg/ml. In other embodiments that may be combinedwith the preceding embodiments, the average blood concentration ofestrogen after continuous use is less than 12 m I.U., less than 10 mI.U., less than 8 m I.U., less than 6 m I.U., less than 5 m I.U., lessthan 4 m I.U., or less than 3 m I.U. In other embodiments which may becombined with the preceding embodiments, the method includes abioadhesive viscoelastic compound for timed release of the estrogen orestrogen analog or other estrogen receptor modulator. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is in a concentration sufficient to achieve thetimed release. In other embodiments which may be combined with thepreceding embodiments that include a bioadhesive viscoelastic compound,the bioadhesive viscoelastic compound is selected from the groupconsisting of polycarbophil, hyaluronate, chitosan, a polysaccharide andpolycarboxylated polymer, carboxylmethyl cellulose, hydroxypropyl methylcellulose, liposomes, a polysaccharide and a positively-chargedsubmicron emulsion. In other embodiments which may be combined with thepreceding embodiments that include a polysaccharide, the polysaccharideis xanthan gum, which optionally can be between about 0.1% to about 1%(w/w) or about 0.6% (w/w). In other embodiments which may be combinedwith the preceding embodiments that include carboxylmethyl cellulose,the carboxylmethyl cellulose is LACRISERT™. In other embodiments whichmay be combined with the preceding embodiments that include apositively-charged submicron emulsion, the positively-charged submicronemulsion is NOVASORB™ or CATIONORM™. In other embodiments which may becombined with the preceding embodiments that include liposomes, theliposomes are multi-vesicular. In other embodiments which may becombined with the preceding embodiments that include multi-vesicularliposomes, the multi-vesicular liposomes are DEPOFOAM™. In otherembodiments which may be combined with the preceding embodiments thatinclude a polysaccharide and polycarboxylated polymer, thepolysaccharide and polycarboxylated polymer is PROLOC™. In otherembodiments which may be combined with the preceding embodiments thatinclude a bioadhesive viscoelastic compound, the bioadhesiveviscoelastic compound is a carbomer, optionally a carbopol, whichoptionally is carbopol is carbopol 980 or carbopol 940. The carbomer maybe from about 0.5% to about 4% or about 2%. In other embodiments whichmay be combined with the preceding embodiments, the composition furthercomprises cyclodextrin, which optionally can be from about 0.1% to about7% (w/w), from about 0.1% to about 1% (w/w), or about 0.4% (w/w). Inother embodiments which may be combined with the preceding embodiments,the timed release comprises a controlled, sustained drug release ratewhich does not vary by more than about 50% for a period of at leastabout 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the timed release comprises a controlled,sustained drug release rate which delivers a dose of between 0.1 pg and1.0 pg of estrogen or the estrogen analog or other estrogen receptormodulator per hour at least about 6-8 hours after application, at leastabout 8-12 hours after application, at least about 12-24 hours afterapplication, at least about 24-48 hours after application, or at leastabout 48-72 hours after application. In other embodiments which may becombined with the preceding embodiments, the timed release comprises acontrolled, sustained drug release rate which delivers as measured by aFranz diffusion cell model with simulated tear flow a dose of between0.1 mg/ml and 1.0 mg/ml of estrogen or the estrogen analog per hour atleast about 6-8 hours after application, at least about 8-12 hours afterapplication, at least about 12-24 hours after application, at leastabout 24-48 hours after application, or at least about 48-72 hours afterapplication. In other embodiments which may be combined with thepreceding embodiments, the method further includes a therapeuticallyeffective amount of an androgen. In other embodiments which may becombined with the preceding embodiments that include an androgen, theandrogen is selected from the group consisting of testosterone,dihydrotestosterone (also termed allodihydrotestosterone,androstanolone, stanolone, 5 alpha-dihydrotestosterone),fluoxymesterone, stanozolol, nortestosterone propionate,dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives. In other embodiments which may becombined with the preceding embodiments that include an androgen, thetherapeutically effective amount of androgen is sufficient to reduceocular inflammation.

Yet another aspect includes use of a topical application pharmaceuticalfor use in either of the two preceding aspects with any of theirrespective combinations of embodiments in the manufacture of amedicament for alleviation of kerato-conjunctivitis sicca (dry eyesyndrome). In certain embodiments, the kerato-conjunctivitis sicca isassociated with post-menopausal subjects, premature ovarian failure,post-operative refractive surgery patients, corneal transplant patientsor patients with other conditions that cause dry eye symptoms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mean change for total subject scores of the test of anon-time release formulation.

FIG. 2. shows the mean change for SPK testing a non-time releaseformulation.

FIG. 3. shows the mean change for the Rose-Bengal testing a non-timerelease formulation.

FIG. 4 shows the mean change for the TBUT testing a non-time releaseformulation.

FIG. 5 shows the mean change for Osmolarity a non-time releaseformulation.

FIG. 6 shows a graph of the AST averaged for both eyes over time for theconcentrations of non-time release formulations tested in the Phase IIa(0.1% and 0.25%) and Phase IIb (0.1% and 0.05%) trials.

FIG. 7 shows a graph of the SPK averaged for both eyes over time for thetwo concentrations of non-time release formulations tested in the PhaseIIb (0.1% and 0.05%) trial.

FIG. 8 shows a graph of the LGS-cornea averaged for both eyes over timefor the two concentrations of non-time release formulations tested inthe Phase IIb (0.1% and 0.05%) trial.

FIG. 9 shows a graph of the AST FBS for both eyes over time for theconcentrations of non-time release formulations tested in the Phase IIa(0.1% and 0.25%) and Phase IIb (0.1% and 0.05%) trials.

FIG. 10 shows a graph of the estradiol eroded solution concentrationover time for the standard formulation (aqueous) and for four noveltimed release formulations.

FIG. 11 shows a second graph of the estradiol eroded solutionconcentration over time for the standard formulation (aqueous) and forfour novel timed release formulations with values on the y-axis cappedat 2.00E-03 to better show differences at lower concentrations.

FIG. 12 shows a graph of the cross-corneal diffusion over time for thestandard formulation (aqueous) and for four novel timed releaseformulations.

FIG. 13 shows a second graph of the cross-corneal diffusion over timefor the standard formulation (aqueous) with the two novel timed releaseformulations tested for 360 minutes.

FIG. 14 shows a graph of the cumulative percent eroded estradiol overtime for the standard formulation (aqueous) and for four novel timedrelease formulations.

DETAILED DESCRIPTION OF THE INVENTION

Time-Release Delivery of Ophthalmic Estradiol Formulations

The ophthalmic estradiol formulations for topical administration to theconjunctiva, the inner eyelid or the outer eyelid described herein maybe conveniently admixed with a non-toxic pharmaceutical organic carrier,or with a non-toxic pharmaceutical inorganic carrier that provides fortimed release of the estrogen or estrogen analog or other estrogenreceptor modulator. These ophthalmic formulations are based upon thesurprising finding that these timed release formulations deliverefficacious doses of estradiol at significantly lower concentrationsover a longer period of time as compared to prior formulations. Thesenew timed release formulations therefore address current needs: thedosing frequency is lower, which can improve patient compliance, and theoverall concentration is lower, which can avoid systemic side effects ofthe estradiol.

Typical of pharmaceutically acceptable carriers are, for example, water,mixtures of water and water-miscible solvents such as lower alkanols oraralkanols, vegetable oils, polyalkylene glycols, petroleum based jelly,ethyl cellulose, ethyl oleate, carboxymethyl-cellulose,polyvinylpyrrolidone, isopropyl myristate and other conventionallyemployed acceptable carriers. The ophthalmic estradiol formulations mayalso contain non-toxic auxiliary substances such as emulsifying,preserving, wetting agents, bodying agents and the like, as for example,polyethylene glycols 200, 300, 400 and 600, carbowaxes 1,000, 1,500,4,000, 6,000 and 10,000, antibacterial components such as quaternaryammonium compounds, phenylmercuric salts known to have cold sterilizingproperties and which are non-injurious in use, thimerosal, methyl andpropyl paraben, benzyl alcohol, phenyl ethanol, oxychlorocomplex-buffering ingredients such as sodium borate, sodium acetates,gluconate buffers, and other conventional ingredients such as sorbitanmonolaurate, triethanolamine, oleate, polyoxyethylene sorbitanmonopailitylate, dioctyl sodium sulfosuccinate, monothioglycerol,thiosorbitol, ethylenediamine tetracetic acid, and the like.

Additionally, suitable ophthalmic vehicles can be used as carrier mediafor the present purpose including conventional phosphate buffer vehiclesystems, isotonic boric acid vehicles, isotonic sodium chloridevehicles, isotonic sodium borate vehicles and the like. The ophthalmicestradiol formulations may also be in the form of a micro- ornano-particle formulation. The pharmaceutical preparation may also be inthe form of a solid insert. For example, one may use a solid watersoluble polymer as the carrier for the medicament. The polymer used toform the insert may be any water soluble non-toxic polymer, for example,cellulose derivatives such as methylcellulose, sodium carboxymethylcellulose, (hydroxyloweralkyl cellulose), hydroxyethyl cellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose; acrylates suchas polyacrylic acid salts, ethylacrylates, polyactylamides; naturalproducts such as gelatin, alginates, pectins, tragacanth, karaya,chondrus, agar, acacia; the starch derivatives such as starch acetate,hydroxymethyl starch ethers, hydroxypropyl starch, as well as othersynthetic derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone,polyvinyl methyl ether, polyethylene oxide, neutralized carbopol (e.g.,carbopol 980 or carbopol 940, in each case from about 0.5% to about 4%(w/w)) and xanthan gum (e.g., about 0.1% to about 1% (w/w)), gellan gum,and mixtures of said polymer. In some embodiments, ophthalmic insertsmay be used for the controlled release of the ophthalmic estradiolformulations described herein. Ophthalmic inserts may be comprised ofhydroxypropyl cellulose, such as the commercially available Lacrisert®(Aton Pharma, Inc.). Alternatively, ophthalmic inserts may comprisepolyvinyl alcohol, as described in U.S. Pat. No. 4,730,013 (Bondi etal.).

In other embodiments, an ophthalmic gel carrier may be used for thecontrolled release of the ophthalmic formulations described herein.Ophthalmic gel carriers can provide good eye retention, can achievesustained release, and are non-toxic. For example, the gel formulationdescribed in U.S. Pat. No. 4,738,851 (Schoenwald et al.) may be used.The ophthalmic gel may be prepared using the combination of sodiumcarboxymethyl-cellulose and colloidal magnesium aluminum silicate, whichavoids an initial burst release.

In yet other embodiments, a cationic emulsion may be used for thecontrolled release of the ophthalmic estradiol formulations describedherein. For example, U.S. Pat. No. 6,656,460 (Benita et al.) describes apositively-charged submicron emulsion containing a phospholipid havingZeta potential values ranging from 34-45 mV and a mean droplet size ofaround 150-250 nm. The resultant electrostatic attraction between thepositively-charged submicron oil droplets in the emulsion and thecorneal eye surface, which is negatively-charged, results in a moreprolonged residence or retention time conducive to topical drug fluxenhancement. Similar ionic or non-ionic emulsions known in the art mayalso be used with the ophthalmic estradiol formulations describedherein.

In an alternate embodiment, it is contemplated that sterile ophthalmicestradiol formulations described herein may be comprised of a liposomaldrug delivery system whose aqueous phase comprises the pharmaceuticalcarrier of the present invention. Liposomal therapy has beensuccessfully used in ophthalmology not only for pre- and postoperativeantisepsis, but also for the treatment of bacterial and viralconjunctivitis and for prophylaxis against ophthalmia neonatorum.(Margalit R., Liposome-Mediated Drug Targeting in Topical and RegionalTherapies, Crit. Rev. Ther. Drug Carrier Syst., 12(2-3):233-61 (1995)).A Method for formulating such a product can be found in U.S. Pat. No.5,662,931, which is herein incorporated by reference. Another example isDepoFoam® (Pacira Pharmaceuticals), which consists of multi-vesicularliposome particles that have numerous internal aqueous chambers thatcontain the encapsulated drug. The multi-vesicular liposome particlescan release the encapsulated drug over a desired period of time, from 1to 30 days.

In another embodiment, a capsule may be used for the controlled releaseof the ophthalmic estradiol formulations described herein. For example,a drug delivery system, such as that described in U.S. Pat. No.7,204,995 (El-Sherif et al.) may be composed of a biodegradablebiocompatible capsule (diameter of 0.01 mm to about 1 mm) associatedwith an estradiol formulation. The drug delivery system capsule wouldencapsulate the estradiol within a polymer shell or sphere. The capsulesmay be stored in the form of a powder that can be suspended in anaqueous carrier solution or dispersed in a gel or an ointment. Thesuspended capsules can be placed into the eye in the form of drops, andthose dispersed in a gel or ointment can be placed in the eye in theform of a gel or ointment. The drug delivery system may work by slowlyreleasing the estradiol formulation into the eye through the polymershell or sphere and/or gets secreted as the polymer degrades. Otherexamples of capsules that may control and maintain the release of theestradiol formulation include U.S. Pat. Nos. 4,853,224; 4,997,652;5,164,188; 5,443,505; and 5,766,242 (Wong, et al.), and are herebyincorporated by reference.

In still another embodiment, hyaluronic acid or hyaluronate may be usedas a viscosity enhancing agent to sustain the delivery of the ophthalmicestradiol formulations described herein. For example, U.S. Pat. No.5,770,628 (Cantoro) discloses an ophthalmic preparation for use as anartificial tear comprising hyaluronate as a viscosity thickener,preferably in the form of an alkali salt and having a molecular weightof 500,000 to 4,000,000 Daltons, preferably about 1,200,000 to 1,400,000Daltons, at a concentration that may range from 0.05 to 2% by weight, aswell as comprising the following minimum quantities of ionic species: 40mmol/l sodium ion, 12 mmol/l potassium ion, 0.4 mmol/l calcium ion, 0.4mmol/1 magnesium ion, 50 mmol/l chloride ion, 7 mmol/l phosphate ionand, preferably, 0.7 mmol/l citrate ion. The preceding size,concentration and quantities are merely for purposes of illustrationwithout being limiting.

In another embodiment, minitablets may be used for the controlledrelease of the ophthalmic estradiol formulations described herein. Forexample, the PROLOC™ (Henkel AG & Co.) bioadhesive minitablets may beused. The PROLOC™ minitablets adhere to the ocular mucosa and remain inplace until fully eroded. PROLOC™ achieves long lasting release and hashigh drug loading.

In still another embodiment, a cyclodextrin may be used for thecontrolled release by itself or in combination with other agentsdisclosed herein. For example, the cyclodextrin may be selected fromα-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin. Each of these threecyclodextrins has been Generally Recognized As Safe by the US FDA.Exemplary ranges for cyclodextrins include about 0.1% to about 10%(w/w), about 0.1% to about 7% (w/w), about 0.1% to about 1.0% (w/w) andabout 0.6% (w/w).

In yet another embodiment, a biodegradable implant may be used for thecontrolled release of the ophthalmic estradiol formulations describedherein. For example, the biodegradable implant may be formulated toprovide a controlled, sustained drug release, as described in U.S. Pat.No. 7,048,946 (Wong et al.). The release rate may be modulated bycombining in the implant hydrophobic and hydrophilic agents. The rate ofrelease of an estradiol formulation may be controlled by the rate oftransport through a polymeric matrix of the implant, and the action ofthe modulator. By modulating the release rate, the estradiol formulationis released at a substantially constant rate, or within a therapeuticdosage range, over the desired period of time. The rate of release maynot vary by more than about 100% over the desired period of time,preferably by not more than about 50%. The release modulator may act toaccelerate or retard the rate of release. Accelerators may bephysiologically inert, water soluble polymers, e.g. low molecular weightmethyl cellulose or hydroxypropyl methyl cellulose (HPMC); sugars, e.g.monosaccharides such as fructose and glucose, disaccharides such aslactose, sucrose, or polysaccharides such as cellulose, amylose,dextran, etc. Alternatively, the accelerator may be a physiologicallyactive agent, allowing for a combined therapeutic formulation. Thechoice of accelerator in such a case will be determined by the desiredcombination of therapeutic activities. Additionally, release retardantsmay be hydrophobic compounds which slow the rate of release ofhydrophilic drugs, allowing for a more extended release profile, and mayinclude non-water soluble polymers, e.g. high molecular weightmethylcellulose and ethylcellulose, etc., low water soluble organiccompounds, and pharmaceutically active hydrophobic agents.

It should be understood that the ophthalmic estradiol formulationsdescribed herein, for example 17-β-estradiol, may be used with any ofthe time-release or controlled release formulations described herein. Insome embodiments the 17-β-estradiol concentration in solution may bepresent in amounts that range from 1%, 3%, or 5%, to 10%, 15%, or 20%.In other embodiments the 17-β-estradiol concentration in solution may bepresent in amounts ranging from 0.1%, 0.3%, or 0.5% to 1%, 3%, or 5%. Instill other embodiments the 17-β-estradiol concentration in solution maybe present in amounts ranging from 0.01%, 0.03%, or 0.05% to 0.1%, 0.3%or 0.5%. In preferred embodiments, the concentration of estrogen orestrogen analog or other estrogen receptor modulator, preferably17-β-estradiol, is between about 0.005% and 5%, about 0.005% and about0.1%, 0.005% and less than 0.05%, 0.01% and about 0.04%, about 0.05% andabout 0.5%, about 0.05% and about 0.1%, about 0.1% and about 5%, orabout 0.5% and about 5%, or is less than or equal to 0.1%weight-by-weight of estrogen or the estrogen analog or other estrogenreceptor modulator.

Micro-Dose Delivery of Ophthalmic Estradiol Formulations

In addition to, or as alternative to, the time-release formulations, theophthalmic estradiol formulations for topical administration to theconjunctiva, the inner eyelid or the outer eyelid described herein maybe formulated for micro-dose delivery. Micro-dose formulations areanother way to improve the concentration of estrogen delivered to theocular tissues. Micro-dose formulations deliver a therapeuticallyeffective dose of the estrogen or estrogen analogs or other estrogenreceptor modulators disclosed in this specification in a lower volumethan the standard ophthalmic drop, which therefore can include a higherconcentration. The lower volume may be delivered by any means availableto one of skill in the art. By way of example, a specializedmicropipette that can deliver smaller than the standard ophthalmic dropmay be used.

By way of example, the formulation disclosed in U.S. Pat. No. 6,096,733of one drop of 0.1% 17-β-estradiol utilizes a standard ophthalmic drop(35 to 50 μl), so the dose is at least 35 micrograms of 17-β-estradiol.A preferred micro-dosing volume would be from 5 to 15 μl which couldhave a higher concentration while still delivering a lower overall doseof less than 35 micrograms. This lowered overall dose will have theeffect of lowering the systemic effects of the drug and increasingadsorption. This method can be used with or without adding the necessaryretention enhancing agents (the timed release/bioadhesive viscoelasticcompounds) as discussed herein.

Ophthalmic Estradiol Formulations

This section sets out exemplary preferred formulations that may be usedas starting points for formulation of the time-released forms disclosedherein. The exemplary starting formulations disclosed in this sectionare not limiting and one of skill in the art may readily begin with anypreferred formulation when generating a timed release form a disclosedherein.

The preferred embodiments prior to formulation in a time-release form isas an aqueous solution of derivatives of estrogen known as17-β-estradiol (or the 3-phosphate disodium salt) and its water-soluble,storage-stable derivatives (β-estradiol glucuronide β-estradiolhemisuccinate, β-estradiol phosphate, β-estradiol sulfate and their 3,17diesters, 17 monoesters and 3 monoesters) and/or one or more androgens,preferably selected from the group consisting of17-α-methyl-17-β-hydroxy-2-oxa-5α-androstan-3-one,4,5α-dihydrotestosterone derivatives, testosterone derivatives,19-nortestosterone derivatives, 17β-hydroxy-5α-androstane derivativescontaining ring A unsaturation, their esters, and their cationic orphosphorylated derivatives, designed to increase solubility inhydrophilic media. Particularly preferred androgens are17-α-methyl-17-β-hydroxy-2-oxa-5α-androstan-3-one,4,5α-dihydrotestosterone and phosphorylated derivatives. Particularlypreferred pharmaceutically active substances for use with thepharmaceutical carrier of the present invention are those that arederivatized to have enhanced solubility and stability at essentiallyneutral pH 6-8 (though the pH is not absolutely critical and couldsuitably range between 4-8.5).

The formulation of 17-β-estradiol (as the 3-phosphate disodium salt) isC₁₈H₂₃O₅P₁Na₂, having a molecular weight of 396.3 (anhydrous). Each gramof 17-β-estradiol (as the 3-phosphate disodium salt) containsapproximately 687 milligram of 17-β-estradiol on an anhydrous basis.17-β-estradiol (as the 3-phosphate disodium salt) is available fromResearch Plus, Inc., Bayonne, N.J. 07002 (catalog No. 1850-5). Thecompound is a white crystalline powder with an ill-defined melting pointand purity better than 98%. The material is to be stored in sealed vialsunder refrigeration when not in use.

In one embodiment, it is contemplated that a sterile, ophthalmicsolution of 17-β-estradiol can be comprised of a liposomal drug deliverysystem (Margalit R., Crit. Rev. Ther. Drug Carrier Syst. 1995;12(2-3):233-61). Liposomal therapy has been successfully used inophthalmology not only for pre- and postoperative antisepsis, but alsofor the treatment of bacterial and viral conjunctivitis and forprophylaxis against ophthalmia neonatorum (Reimer K, et al., Dermatology1997; 195 Suppl. 2:93-9). A Method for formulating such a product can befound in U.S. Pat. No. 5,662,931 (Munechika, K. et al.) and hereinincorporated by reference. A liposome system for delivery of17-β-estradiol is disclosed in example 3 below.

In an alternate embodiment, a sterile, ophthalmic suspension of17-β-estradiol cypionate is dissolved to form a 0.1% (by volume)solution in a vehicle which may in one embodiment take the form of alipid based solution having a pH within the range of 4-8 with apreferred range of about 6-8.

In another alternate embodiment, a sterile, ophthalmic solution of17-β-estradiol (as 3-phosphate disodium salt) may be dissolved to form a0.1% (by volume) solution in a vehicle which may in one embodiment takethe form of a typical over-the-counter artificial tear solution. Theconcentration of 17-β-estradiol in the vehicle may be increased ordecreased depending on the activity of the 17-β-estradiol (as3-phosphate disodium salt). Below are alternate embodiments of thedrops.

A. 17-β-estradiol (as the 3-phosphate disodium salt) and itswater-soluble, storage-stable derivatives (beta-estradiol glucuronide,beta-estradiol hemisuccinate, beta-estradiol phosphate, beta-estradiolsulfate and their 3,17 diesters, 17 monoesters and 3 monoesters). The17-β-estradiol 3-phosphate disodium salt may be employed in thepreferred embodiment because of the enhanced solubility and stability ofthe particular derivative at essentially neutral pH 6-8 (though the pHis not absolutely critical and could have a pH between 4-8.5) and theease of sterile ophthalmic manufacture.

TABLE 1 B. A sterile ophthalmic ointment formulated to melt at bodytemperature containing: Compound Concentration (w/v %) 17-β-estradiol(microcrystalline) 0.05-0.1  propyl paraben (USP) 0.2 anhydrous liquidlanolin 5.0 mineral oil (USP) 10.0  white petrolatum (USP) 84.6-84.7

TABLE 2 C. A sterile aqueous ophthalmic suspension formulated tocontain: Compound Concentration (w/v %) 17-β-estradiol(microcrystalline) 0.05-0.1 polysorbate 80 (USP) 0.2 povidone (USP)(K-30 type) 1.0 hydroxyethylcellulose (USP) 0.5 sodium chloride (USP)0.5 disodium edate (USP) 0.05 benzalkonium chloride (USP) 0.005 dil. HCLfor pH adjustment qs purified water (USP) qs

The following is a description of the manufacturing and packagingprocedure for a preferred drug product of the invention. Moreinformation on the preparation and characteristics of poly-estradiolphosphate is set forth in the article by E. Diczfalusy entitled HighMolecular Weight Enzyme Inhibitors, pp. 1675-1689, Chemica ScandinaviaVol. 12 (1958) No. 8, which is incorporated herein by reference.

The method of synthesis of 17-β-estradiol 3-phosphate disodium isreported in Acta Chem. Scan. 12, 1675-1689 (1958) and is brieflydescribed as follows:

17-β-estradiol 17-acetate (Molecular Weight=314.4, Melting Point220-224° C. and optical rotation 47°) may be phosphorylated in thepresence of concentrated ortho-phosphoric acid (H₃PO₄) with heat andrefluxing to yield the intermediate 17-β-estradiol 3-phosphate17-acetate. The latter compound may be selectively hydrolyzed in thepresence of sodium bicarbonate in aqueous alcohol to yield sodiumacetate and 17-β-estradiol 3-phosphate disodium. The desired steroidphosphate ester may be recrystallized from dilute alcohol.

A complete list of components that can be present in a preferredembodiment of the drug product in accordance with the presentinvention—including the drug substance, is as follows (in percentages byvolume):

-   -   17-β-estradiol (as 3-phosphate disodium salt) 0.1%    -   The concentration in subsequent batches may be increased or        decreased depending upon the activity of 17-β-estradiol (as        3-phosphate disodium salt).

The vehicle may be supplied as a typical over-the-counter artificialtear (solution) with a composition of the vehicle as follows:

TABLE 3 Compound Concentration povidone (USP) (K-30 type) 1.67% byvolume  hydroxyethylcellulose (USP) 0.44% by volume  sodium chloride(USP) 0.6% by volume anhydrous sodium phosphate 0.3% by volume (Na₂HPO₄)(USP) disodium edate (USP) 0.1% by volume dil. HCL or NaOH for pHadjustment qs purified water (USP) qs

Based upon the chemistry of steroid phosphate esters, clarity of aqueoussolution at essentially neutral pH values should be indicative of thepresence of intact steroid phosphate ester. On the other hand,turbidity, haze formation or precipitate formation would indicate thepresence of hydrolyzed, insoluble, free 17-β-estradiol.

Very small amounts of free, water-insoluble 17-β-estradiol can betolerated in the steroid ophthalmic product as long as the homogeneityof the drug product is maintained because it is the 17-β-estradiolitself that is undergoing clinical study and not the phosphate esterpro-drug.

Solutions of drug product are preferably stored at controlled roomtemperature (15 to 30° C.) preferably at 22 to 24° C. as long asadequate physical stability (i.e., clarity of solution) is maintained.Otherwise storage under refrigeration (less than 10° C.) may berequired.

It is also contemplated in an alternate embodiment, that the above dropsmay use a modified preservative system as described in Table 3. Moreparticularly, this alternate embodiment can use methyl paraben at aconcentration (w/v) of about 0.05-0.5% in combination withphenoxyethanol at a concentration (w/v) of about 0.1-1.0%.

The placebo that can be used in controlled clinical trials is thevehicle that can be used in the manufacture of the drug product, namelya typical over-the-counter artificial tear (solution), similar to theformula of which is identified previously. The placebo may be anon-prescription, over-the-counter drug product used to providetemporary relief of dry eye symptoms. It may contain mucin-likesubstances (povidone and hydroxyethylcellulose) which mimic the actionof the conjunctival mucus or render the surface of the eye morewettable. The vehicle may help keep the eye moist and assures that thetear film can spread easily and evenly over the eye surface.

The preferred vehicle for 17-β-estradiol (as 3-phosphate disodium salt)may have the following attributes:

-   -   a) a sterile, buffered isotonic or hypotonic solution.    -   b) contains mucin-like substances that tend to increase the        contact time between the active drug substance (17-β-estradiol        (as the 3-phosphate disodium salt) and the eye surface.    -   c) free of benzalkonium chloride, which is a cationic surfactant        that is known to be incompatible in solutions with steroid        sodium phosphate salts.

The following quality control procedures may be employed to assureidentity, strength, quality and purity of the drug product:

Representative samples of finished drug product may be opened andexamined for clarity of solution (clear, colorless to pale yellowsolution, essentially free of foreign matter), pH content (not less than4 and not more than 8.5) and a simple potency assay (absorbance read at280 nanometers using 1 centimeter cells in a suitable spectrophotometerafter diluting the drug product with alcohol or methanol to a suitableconcentration.

In an alternate embodiment, it is contemplated that the composition ofsaid invention may be free of any preservative compounds such as, forexample, Thimerosal (USP) and said invention may be provided to patientin a sterile single or similar package allowing no more than 3 to 5 daysof use before the patient discards the package.

In another alternate embodiment, it is contemplated that the presentinvention may utilize an ocular insert means of delivering the17-β-estradiol ingredients directly to the ocular surface andconjunctiva. Such delivery systems are well known in the art and areexemplified by U.S. Pat. No. 4,478,818 (Shell et al.) of AlzaCorporation (Palo Alto, Calif.) and hereby incorporated by reference.

In yet another alternate embodiment, it is contemplated that the presentinvention may utilize a thermosetting gel with a low sol-gel transitiontemperature as a method of delivering the 17-β-estradiol ingredientsdirectly to the ocular surface and conjunctiva. Such delivery systemsare well known in the art and are exemplified by U.S. Pat. No. 4,474,571(Haslam et al.) of Merck & Co., Inc. (Rahway, N.J.) and herebyincorporated by reference.

The quality control procedures are also the same as for the active drugproduct described above with the exception that the ultravioletabsorbance at 280 nanometers of the placebo solution when diluted to thesame concentration as the active drug product will fail to indicate thepresence of 17-β-estradiol (as the 3-phosphate disodium salt) inrepresentative samples of the placebo solution.

Ophthalmic Androgen Formulations

Regarding the androgen that may be included with the estrogen orestrogen analog or other estrogen receptor modulator, selection of themost appropriate therapeutic androgen will depend upon a given hormone'simmunological activity, potential side effects and form ofadministration. For example, topical testosterone may be quite effectivein reducing ocular surface inflammation, and its methylated analogueappears to have no toxic side effects on parameters such as intraocularpressure (Knepper, P. A., Collins, J. A., and Frederick, R., Effect ofDexamethasone, Progesterone, and Testosterone on IOP and GAGs in theRabbit Eye, Invest. Ophthalmol. Vis. Sci., 26:1093-1100 (1985)).However, a variety of other modified and/or anabolic androgens (Wilson,J. D., and Foster, D. W., eds., “Williams Textbook of Endocrinology,” WBSaunders Company, Philadelphia (1985), Vida, J. A., “Androgens andAnabolic Agents,” Academic Press, New York (1969)) may be more effectivethan testosterone.

In order to increase the aqueous solubility of the androgen,phosphorylated ester derivatives of the androgens are preferred and canbe prepared by means commonly available in the art. For example, themost convenient method of synthesis of steroid esters is reaction of thesteroid in a 2:1 mixture of pyridine and the anhydride of the desiredester: for example, propionic anhydride would be used to make thepropionate ester. A large excess (at least 10 times) of the anhydridecompared to the steroid would be required. This would then be purifiedby diluting with at least 10 parts of water to each part of pyridine,adding 1 part ether, decanting the water after shaking, and then washingwith 10 parts water repeatedly in a separatory funnel. This would befollowed preferably by recrystallization or chromatography forpurification.

Androgens that may be used include testosterone, dihydrotestosterone(also termed allodihydrotestosterone, androstanolone, stanolone, 5alpha-dihydrotestosterone), fluoxymesterone, stanozolol, nortestosteronepropionate, dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone), oxandrolone; methyldihydrotestosterone (alsotermed methylandrostanolone), oxymetholone, 5alpha-androstan-17β-ol-3-oxime, 5 alpha-androstan-17alpha-ol-3-one-acetate, (1) 2,(5 alpha)-androsten-17β-ol, 5alpha-androstan-2 alpha-methyl-17β-ol-3-one, methyltestosterone, andtheir soluble ester derivatives.

These androgens are representative of the major structural subclasses ofandrogens, as disclosed in Vida (Vida, J. A., “Androgens and AnabolicAgents,” Academic Press, New York (1969), hereby incorporated byreference. The subclasses include (a) androgenic compounds with unusualstructural features (e.g., 17 alpha-methyl-17-β-hydroxy-2-oxa-5alpha-androstan-3-one, also termed oxandrolone); (b) testosteronederivatives (e.g., methyltestos-terone); (c) 4,5alpha-dihydrotestosterone derivatives (oxymetholone); (d) 17β-hydroxy-5alpha-androstane derivatives containing a ring A unsaturation, excludingtestosterone derivatives (e.g., 2,(5 alpha)-androsten-17β-ol); and (e)19-nortestosterone derivatives (e.g., 19-nortestosterone propionate). Itmay be that certain structural features impart more optimalimmunosuppressive characteristics, which would be of benefit inselecting specific androgens for human use.

Also, relative to standards (typically testosterone), these androgensmay include compounds displaying: (a) augmented androgenic (i.e.,virilizing) activity coupled with an even larger increase in anabolicactivity (e.g., fluoxymesterone); (b) enhanced anabolic action withunchanged androgenic effects (e.g., oxymetholone, dihydrotestosterone);(c) decreased androgenic ability with unchanged anabolic activity (e.g.,19-nortestosterone propionate); and (d) decreased androgenic capacityparalleled by increased anabolic activity (e.g., oxandrolone,stanozolol). Preferred androgens for use in compositions of theinvention are those which have far more anabolic, than virilizingeffect, (e.g., oxandrolone possesses 322% of the anabolic and 24% of theandrogenic activity of methyltestosterone (Vida, J. A., “Androgens andAnabolic Agents,” Academic Press, New York (1969)).

Additional Ophthalmic Carrier Formulations

This section sets out additional exemplary preferred formulations thatmay be used as starting points for formulation of the time-releasedforms disclosed herein. As above, the exemplary starting formulationsdisclosed in this section are not limiting and one of skill in the artmay readily begin with any preferred formulation when generating a timedrelease form a disclosed herein. In some embodiments, the startingcarrier composition prior to formulation in a time-release form oraddition of the mucoadhesive viscoelastic compound may be an aqueoussolution comprising, on a weight percent basis, about: Dibasic sodiumphosphate, USP 0.05-1.0% Sodium Chloride, USP 0.2-0.9% Edetate disodium,USP 0.05-1.0% Povidone, USP 0.05-2.0% Poloxamer NF 0.001-0.05%Polyethylene glycol 0.05-1.0% Hydroxyethyl Cellulose NF 0.05-1.0%Purified water, USP, q.s to 100% HCl or NaOH to adjust pH to pH 6-8.

A more preferred starting composition of the invention may comprise:Dibasic sodium phosphate, USP 0.3% Sodium Chloride, USP 0.6% Edetatedisodium, USP 0.1% Povidone, USP 0.37% Poloxamer NF 0.004% PEG 0.12%Hydroxyethyl Cellulose NF 0.2% Purified water, USP, q.s to 100% HCl orNaOH to adjust pH to pH 6-8.

The preferred Povidone can be K-15, or K-17, with K-17 beingparticularly preferred. The preferred Poloxamer can be Poloxamer 188.The preferred polyethylene glycol can be PEG 3350, and the preferredhydroxyethyl cellulose is Hydroxyethyl Cellulose 100.

The composition may optionally comprise one or more preservatives suchas methylparaben, NF, and/or propylparaben, NF, and/or phenoxyethanoleach of which may be present in an amount ranging from about 0.005 toabout 0.5% by weight. The preferred composition can comprise both 0.04weight percent methylparaben and 0.02 weight percent propylparaben.

The foregoing ophthalmic carriers are merely for illustrative purposeswithout limiting the scope of the invention. One of skill in the art mayreadily utilize any other ophthalmically suitable carrier as long as thecarrier is compatible with the estrogen or estrogen analog or otherestrogen receptor modulator and with the time-release component(s) ofthe formulation.

The amount of active ingredient that can be admixed with the chosencarrier may depend on the use and factors such as the age of thepatient, the particular condition to be treated, the frequency ofadministration, and the means of administration. The concentration ofactive ingredients can range from about 0.001 percent to about 10percent by weight. In a most preferred embodiment, the concentration of17-β-estradiol 3-phosphate disodium may be in the range of 0.01-1.0weight percent. In another preferred embodiment, an androgen may bepresent in a concentration of about 0.001 to about 0.1 percent by weightof the composition.

The pharmaceutical carrier may itself be useful in alleviating symptomsof dry eye syndrome. As such it may be used by itself as a placebo, atear substitute, or otherwise with or without the presence of activeingredients. The carrier, without the active ingredients, may also beuseful in alleviating discomfort and minor irritation associated withthe wearing of contact lenses.

EXAMPLE 1 Initial Dose Studies

Although the present invention has been described with reference toseveral illustrative examples, it will be understood that the inventionis not limited to the examples given herein by way of illustration, butonly by the scope of the appended claims.

Prior to an application of a drug formulated in accordance with thepresent invention it was necessary to establish the presence of dry eyesyndrome in the test population and to follow its course undertreatment. It is imperative that the diagnosis of dry eye syndrome becorrect and the patient not be suffering from other or additionalophthalmic diseases such as Sjörgen's syndrome.

The diagnosis of dry eye syndrome in the present invention, was made onthe basis of the following tests. Initially, microscopic evaluation ofthe tear film with particular attention to the marginal tear strip,viscosity and debris content of the precorneal tear film, and lidexamination is performed. This is followed by staining the ocularsurface with Rose Bengal, a vital dye which indicates cellular damage,Schirmer testing, tear osmolarity, measurement of tear break-up time(TBUT), and finally, the maturation index (a Papanicolaou stained sampleof conjunctival epithelium) are then performed.

The diagnosis of menopause was confirmed with follicular stimulatinghormone and luteinizing hormone serum determinants. Dry eyepostmenopausal females had mean E₂ (estradiol levels) of 3.47picograms/milliliter. Normal postmenopausal females had mean E₂(estradiol levels) of 16.05 picograms/milliliter (U.S. Pat. No. Re.34,578, col. 2, Ln. 56-59).

A non-time release topical drug product comprised a sterile solution ofestradiol cypionate dissolved in a lipid (oil-based) vehicle at aconcentration of 0.05 milligram/milliliter was tested for itseffectiveness as a therapy for postmenopausal, dry-eye syndrome in acontrolled, double-blind study. The non-time release topical drugproduct was tested to determine dose response. As a pilot project, thedose was changed after one week to 0.1 milligram/milliliter, and aftertwo weeks to 1.0 milligram/milliliter, all performed as medication inone eye and placebo (medium) in the other.

Two drops given three times a day were indicated, but it was found thatapplication may be more or less frequent. However, it was determinedthat other alternative pharmaceutical modes of administration may beused—such as a slow release mode, or any other topical method, and thatthe concentration may vary with individual response, as well as thetreatment intervals and duration. Blood levels of the hormone used werealso determined. A control bottle of just the aqueous vehicle was alsomade, using the estrogen preparation for one eye of the patient and thecontrol vehicle for the other eye. A dosage of the drops four times aday for several weeks, during which time osmolarity and maturationindices were performed. No change was noted in the maturation index orosmolarity, thus the concentration was increased to (0.05%). After tendays of treatment, the control eye showed no change in the experimentalparameters while the eye receiving topical estrogen showed epithelialmaturation commonly seen during ovulation in premenopausal females.

The effectiveness of estrogen and its derivatives in treatingkeratoconjunctivitis sicca was confirmed by the use of an intravenoussterile solution of conjugated estrogens kept refrigerated during use.The drug remained active for 60 days when refrigerated at 4-15° C.

Blood Concentrations of Estrogen in Subjects Treated with 0.1%17-β-estradiol were measured. The measurements fell within a range 0 to30 milli-International Units (m I.U.) of 17-β-estradiol.

TABLE 4 Estrogen Blood Level at 0.1% dose Patient No. Estrogen BloodLevel at 0.1% dose 1 13 2 36 3 136 4 3 5 9 6 40 7 19 8 89 9 12 10 22 1141 12 44

The average blood concentration of estrogen in test subjects was 15.5 mI.U. The average blood concentration of estrogen in the placebo groupwas 0.99 m I.U. at the beginning of the study and 7.34 m I.U. at the endof the study.

EXAMPLE 2 Results of Clinical Trials Using 17-β-Estradiol to Teat DryEye

A phase 1 human clinical trial was conducted between November 1993 andDecember 1995. The objective of the present study was to evaluate theefficacy and safety of a non-time release formulation of 17-β-estradiolin relieving the signs and symptoms of dry eye syndrome inpostmenopausal women. 45 completed subjects were studied in thesingle-center, randomized, double-masked, parallel, placebo controlledstudy. The trial was conducted as follows:

The initial visit (Visit 1) occurred 7 days prior to the actual start ofthe study, where subjects were screened. Information regarding health,ophthalmic history, and patient evaluation and inclusion/exclusion basedon FDA approved criteria were performed. A complete ophthalmicexamination which included:

Acuity testing

Biomicroscopy

Intraocular pressure (IOP)

Ophthalmoscopy

Color vision

Tear osmolarity

Schimer's test

Tear film break-up time (TBUT)

Superficial punctate keratitis (SPK)

Rose bengal staining (RBS)

In addition, blood was drawn for FSH, LH and estradiol levels and aurine sample was collected for pregnancy testing where necessary.Subjects were given the placebo vehicle and instructed to instill thedrops four times a day in both eyes for seven days. Daily diaries wereprovided and subjects were given instructions on using them. They werealso instructed to discontinue use 12 hours prior to the next visit andto do the same before subsequent visits.

On Visit 2 (Day 1 of the study) the patient's medical history wasupdated and all symptoms of ocular discomfort were evaluated andrecorded. The same measurements were taken as in Visit 1. All thosewhich qualified were randomized and received 0.1% estradiol, 0.25%estradiol or placebo.

The first drop of the assigned study drug or placebo was instilled inthe eyes of each subject at the investigator's office. Signs or symptomsof dry eye were then evaluated by the investigator at 15, 30, and 60minutes post-instillation. Subjects were then instructed to begininstilling the drug four times a day for a total of 90 days beginningthe next day. Signs and symptoms of dry eye were evaluated by subjectsimmediately before the 8:00 a.m. (first) dosage and 8:00 p.m. (last)dosage of the drops.

The subjects returned for follow-up on days 14, 30, and 60. During thesevisits the patient's medical history was updated and all symptoms ofocular discomfort were evaluated and recorded. The same measurementswere taken as in Visit 2.

On the final exit visit (Visit 6), the subjects stopped the treatmentregimen and reverted back to their usual ocular lubricants. The sameexamination procedures were performed as on Visit 1. All study drugs andsubject daily diaries were collected and an exit form for each patientwas completed for all subjects. All subjects were advised to return fora post-study follow-up on day 105 (Visit 7), with the same examinationprocedures were performed as on Visit 1.

The results of the study were most impressive. The data indicated astrong trend that estradiol drops significantly improve both hallmarksymptoms and objective parameters. Graphical representations of the meanchange for subjective scores, SPK, Rose Bengal, TBUT, and Osmolarity areshown in FIGS. 1 to 5, respectively. Statistically significant resultswere found when evaluating subjective complaints of redness and foreignbody sensation amongst placebo and treatment groups. When comparing thetwo treatment groups combined vs. placebo, the p-values for tear breakup time were 0.08 and for the subjective measures, the p-value was 0.13.When the 0.1% group was independently compared to placebo, the p-valuefor osmolarity came to 0.056 and for tear break up time 0.066.

Forty four of forty five patients completed the entire study. Althoughthe study size is limited (due to severe inclusion/exclusion criteria)and provides us with only a 40% chance of finding a moderate to largeclinical effect at a p=0.05, several findings stand out. If one looks atthe means of the differences for all groups (placebo, 0.1% Estradiol,0.25% Estradiol), the mean difference in the score between the secondand sixth visits (T6-T2) for all subjective (FIG. 1) and objectiveoutcome' variables), several of the measures (tear break up time (FIG.4), Rose Bengal (FIG. 3), Osmolarity (FIG. 5), the sum of the subjectivemeasures) deviate in the hypothesized direction. For example, the meansfor tear break up time decrease with increasing dosage. The second andsixth visits were used because the study medication was started on visit2 and stopped at visit 6.

A Wilcoxon Rank Sum Test on the 0.1% group was independently comparedwith the placebo group on all clinical and subjective measures (table5). As this data was normally distributed, t-tests were used to assessif the means of the variables differed.

TABLE 5 Analysis Of Mean Difference Between Placebo And 0.10% EstradiolTreatment Test N value P-value Osmolarity 0.056 Placebo 12 0.10% 18 TBUT0.066 Placebo 12 0.10% 18 Subjective 0.17 Placebo 12 0.10% 18 RoseBengal 0.17 Placebo 12 0.10% 18 SPK 0.72 Placebo 12 0.10% 18 Schirmer's0.82 Placebo 12 0.10% 18

Finally, an analysis of the individual subjective ratings was performed(FIG. 1). Three tests were conducted. First, an overall test looked fordifferences in the distribution of scores among the three groups withoutregard to level. Differences between the 0.1% group and the placebogroup were then tested (Table 6). Two ratings emerged as significant ata p-level of less than 0.05: foreign body sensation and redness. Forboth ratings, the treatment groups showed significantly more improvementthan the placebo group.

TABLE 6 Analysis of Subjective Scores of Placebo v. 0.10% Estradiol TestWilcoxon values FBS 0.044* Redness 0.013* Itching 0.20 Burning 0.85*indicates that the results were statistically significant

The FDA found that the results of the study succeeded in its purpose indemonstrating the effectiveness of estrogen eye drops in thepostmenopausal dry eye and approved advancing the study to a phase 3trial involving two formal clinical trials in two separate centers usingthe final formulation.

A small Phase IIb trial was conducted to compare the 0.1% formulationwith a 0.05% formulation to see if a lower dose could be used to obtainsimilar results while lowering the systemic estradiol levels asindicated on Table 4. The Target Patient Population (TPP) group wasdefined as those patients enrolled who presented with an AST score of <7mm/5 minutes and an LGS-Conjunctiva (nasal+temporal) score of >2 (≧3) inthe Signal Eye on Day 0. The TPP was the patient group withmoderate-to-severe Dry Eye that will likely be selected for the PhaseIII trial. As opposed to the ITT (Intent-to-Treat) group which comprisesall enrolled patients, the TPP was a subgroup that comprises about half(15/30 in the 0.1% group) of the ITT, and which demonstrated the mostbenefit in the main signs (AST (Anesthetized Schirmer's Test—a test forthe volume of tears produced in 5 minutes); LGS-cornea (Lissamine GreenStaining—a test for damage to the cornea and conjunctival surface); andSPK (Superficial Punctate Keratitis—similar to LGS but using a differentdye)) and symptom (FBS (Foreign Body Sensation—typical symptom in dryeye found in many patients)).

The results for the Phase IIb trial showed that the lower concentrationwas generally less efficacious that the 0.1% formulation. For AST, the0.1% formulation was clearly superior to the 0.05% formulation and thevehicle alone, which produced similar results. See FIG. 6. This appliedfor study eye alone and both eyes averaged data. For SPK, the 0.1%formulation was superior to the vehicle alone (though not statisticallysignificant in single eye evaluation presumably due to small sample sizeand variability of individual data points but statistically significantwhen both eyes averaged), and the effect was numerically better than the0.05% formulation and occurred earlier. See FIG. 7. For LGS, the trendobserved favored the 0.1% formulation over the vehicle alone. See FIG.8. For the FBS, an unexpected reversal for the symptom was observedwhere the 0.05% formulation outperformed the 0.1% formulation. The mostlikely explanation was a combination of the symptoms being subjectiveand therefore subject to a wider variability and the small group size.FIG. 9 shows combined results from the Phase IIa and Phase IIb trials.From the figure, it is clear that the patients receiving the 0.1%formulation in the Phase IIb reported an unusually low response. Thepatients receiving the 0.1% formulation in the Phase IIa reported asimilar response as the patients receiving the 0.05% formulation in thePhase IIb trial.

The overall picture leads to the conclusion that despite the result forFBS, the 0.1% aqueous formulation is the better dose to take to PhaseIII if the formulation were to remain unchanged. The FBS result was theonly one that favored the 0.05% formulation, which may have been due tothe small population size or the subjective nature of the test.

EXAMPLE 3 Formulation of Time-Release Topical Application Pharmaceutical

Various pharmaceutical compositions will be formulated based upon thefollowing starting composition:

TABLE 8 Compound Concentration (w/v %) 17-β-estradiol (microcrystalline)Desired amount polysorbate 80 (USP) 0.2 povidone (USP) (K-30 type) 1.0hydroxyethylcellulose (USP) 0.5 sodium chloride (USP) 0.5 disodium edate(USP) 0.05 benzalkonium chloride (USP) 0.005 dil. HCL for pH adjustmentqs purified water (USP) qs

The formulations will not include any preservative. The followingbioadhesive viscoelastic compounds will be added each in its ownformulation: Sodium Hyaluronate (0.05% to 0.2%), xanthan gum,hydroxypropyl methyl cellulose (0.5% to 2.0%) and polycarbophil. Afteraddition of the bioadhesive viscoelastic compound has been added, theexcipients will be adjusted to maintain isotonicity of the formulation.After adjustment of the isotonicity, the formulations will be subject tobasic diffusion studies to verify adequate delivery of the17-β-estradiol. The concentration of the 17-β-estradiol and/or theamount of bioadhesive viscoelastic will be adjusted such that theappropriate concentration of 17-β-estradiol is delivered. The finalformulations will then be subject to accelerated stability studies forone month at 55° C., 40° C. and 25° C.

EXAMPLE 4 In Vitro Erosion Diffusion Model

After formulations for multiple time-release topical applicationspharmaceutical have been finalized as per Example 3, the time-releaseformulations will be tested in an erosion/diffusion model. Samples willbe pulled from the receiver chamber at selected time points. Sampleswill also be pulled from the outflow of the upper/donor chamber at thesame time points. The samples will be stored and tested by forEstradiol.

EXAMPLE 5 In Vivo Testing

The preferred formulation identified in Example 4 (or in Example 7) willbe made using radiolabeled estradiol for preclinical in vivo animaltesting. Various treatment levels of estradiol for the formulation(s)will be dosed for 7 days at various dosing regimens times per dayfollowed by a radiolabeled treatment on day 8. Samples collected atvarious intervals post-dose of radiolabel.

The plasma, tear, and tissues samples will be tested by liquidscintillation counting to assess recovery of 3H-estradiol in the samplesand provide time-concentration data.

Thus, there will be an adequate number of rabbits for samples at eachtime point for each treated group. The three untreated animals will becollected amongst the treated groups as a check for cross contamination.Total tear weight will be collected. Additional ocular tissue samplesand blood samples will be collected. All tissue samples will be storedon dry ice and then at −80° C. until analysis. All blood samples will bedrawn with EDTA coated syringes. Blood samples will be appropriatelystored to retain integrity prior to analysis.

EXAMPLE 6 Clinical Trial of Timed Release Formulations

The overall design of the phase II clinical trial to test the efficacyof the optimal formulation determined from Example 5 is as follows. Aninitial patient pool of 105 patients will be screened. After a run-inperiod, patients will be randomized to receive one of the test productsor their vehicle. The new formulation will be tested when used as a oncenightly or twice daily application. Table 9 contains additional detailsof the phase II clinical trial.

TABLE 9 Title iDESTRIN ™ New Timed Release Formulation (iDNF) vs placeboProtocol Number ALT-001v1 Development Phase II Primary Objectives 1)iDNF Conc. X % vs iDNF Conc. 2X % vs. iDNF  placebo  i) used ONCE atnight and ii) used TWICE daily. 2) Local Tolerance and systemic safetyof both  iDNF concentrations. 3) Systemic estradiol levels at bothconcentrations  and dose regimens. Secondary Objective 1) Comparisonbetween o.n. and b.d. dose regimens 2) Study Design Double masked,parallel group, 3-arm, placebo controlled randomized study with a run-inperiod. Number of Subjects (3 × 30) + 15 reserve = 105 patients. Numberof Centers 6 sites #pt/site 17 Inclusion Criteria Post menopausalfemales NOT using HRT with moderate to severe dry eye disease. ExclusionCriteria To be defined Duration of study/pt 15 weeks # visits/pt 8(Screening + 7 visits) Test product iDNF Concentration X iDNFConcentration 2X Comparator iDNF vehicle Primary Endpoints Symptoms:OSDI/DEQ Signs: Staining (Oxford Scale) Secondary Individual symptomsincluding ocular discomfort, Endpoints dryness, foreign body sensation,irritation, etc. Signs including Schirmer's test, TBUT, Lissamine greenstaining, tear osmolarity, etc.

EXAMPLE 7 Franz Diffusion Cell Model

A Franz diffusion cell model was modified to include simulated tear flowto better assess the impact of formulation changes on the diffusion ofactive moieties across isolated rabbit corneas. The methodology haspreviously been described (Rowe T E et al., Measurement and Predictionof Timolol Diffusion with and Without Simulated Tear Flow, Associationfor Research in Vision and Ophthalmology Congress, May 2010, Poster2452.). To test this model, an in vitro trans-corneal diffusion studycomparing Timoptic (solution) and Timoptic XE (gel) was performed underboth static and simulated tear flow conditions. The amount of Timololthat diffused across the cornea as well as the amount of Timololretained in the pre-corneal (donor) area were compared. The Franzdiffusion cell model without simulated tears showed no differencebetween Timoptic (solution) and Timoptic XE (gel). By contrast, theresults from the Franz diffusion with simulated tears were comparable tothose published in vivo data for the two products (Burgalassi et. al.(2000) Xyloglucan as a Novel Vehicle for Timolol: Pharmacokinetics andPressure Lowering Activity in Rabbits, J. Ocular Pharm. Therapeutics,16(6): 497-509.). Therefore, the Franz diffusion model with simulatedtears used in this Example 7 provides an accurate model of the deliveryof active ingredients in vivo.

Formulations

In all experiments in this Example 7, the aqueous portion of theformulation was the same as the formulation used in the phase IIb studydiscussed in Example 2 above. The aqueous formulation containing 0.1%17-β-estradiol phosphate was used as control. Test formulations variedin 17-β-estradiol free acid concentration and excipients and include:

-   -   1. Standard formulation (0.078% 17-β-estradiol free acid)    -   2. 0.034% 17-β-estradiol in carbopol 980 (2% w/w)    -   3. 0.025% 17-β-estradiol in carbopol 980 (2% w/w) and hydroxy        propyl beta cyclodextrin (CD) (0.4% w/w)    -   4. 0.017% 17-β-estradiol in Xanthan gum (0.6% w/w)    -   5. 0.016% 17-β-estradiol in Xanthan gum (0.6% w/w) with CD (0.4%        w/w)

Results

The data was obtained by performing three studies. The formulationscompared in each of the three studies were as follows:

Study 1 was a comparison of the following formulations for 180 minutes:

-   -   Standard formulation (the formulation used in the Phase IIb        studies which contains 0.078% 17-β-estradiol phosphate)    -   0.034% 17-β-estradiol in carbopol    -   0.025% 17-β-estradiol in carbopol and cyclodextrin (CD)

Study 2 was a comparison of the following formulations for 180 minutes:

-   -   Standard formulation (the formulation used in the Phase IIb        studies which contains 0.078% 17-β-estradiol phosphate)    -   0.017% 17-β-estradiol in Xanthan gum    -   0.016% 17-β-estradiol in Xanthan gum with CD

Study 3 was a comparison of the following formulations for 360 minutes:

-   -   Standard formulation (the formulation used in the Phase IIb        studies which contains 0.078% 17-β-estradiol phosphate)    -   0.016% 17-β-estradiol in Xanthan gum with and cyclodextrin (CD)    -   0.034% 17-β-estradiol in carbopol

Results—Objective #1

The first objective was to improve the overall availability of17-β-estradiol to ocular surface tissues by increasing the duration ofthe drug on the ocular surface. In other words, allowing the activeingredient to be present on the ocular surface for a longer period oftime.

The impact of the formulation on this parameter was best exhibited bythe concentration profile exhibited in the erosion data. Overall, thedata indicated that delivery of the aqueous (Phase IIb—0.1%17-β-estradiol) formulation to the simulated ocular surface results in asurge of 17-β-estradiol which is rapidly depleted after the firstfifteen minute time interval. The timed release formulations have a morelimited early release profile and maintain a higher concentration on thesurface of the eye until about the 180 minute mark. FIG. 10 is a plot ofthe erosion concentration data.

The standard 0.1% aqueous formulation demonstrated a typical pattern ofsolution erosion with 74% of the erosion occurring within the first 15minutes. This corresponds well with the recorded peak bloodconcentration of estradiol recorded in clinical trials with iDESTRINdiscussed in Example 2 above, which occurred at twenty minutes postinstillation.

Of the novel formulations, only the Xanthan gum P3 formulation, with acorresponding value of 54%, showed similar rapid erosion in theimmediate period post instillation. This formulation appears to minorthe performance of the standard throughout the observation period.

Due to the considerable drop in the 17-β-estradiol concentration fromthe initial data for the standard and the xanthan P3 formulation, it isnot possible to display the changes over time with the otherformulations on FIG. 10. FIG. 11 applies a limit on the upper range ofthe y axis concentration value to 0.0005 mg/ml in order to have animproved review of the differences in concentrations of the differentformulations over a more prolonged time (30 min to 180 min). Thisextended time in estradiol erosion concentration is expected to resultin improved adsorption of the drug by ocular surface tissues.

The key formulation improvements here were observed with the CP3(Carbopol gel formulation) and the Xanthan+CD formulation.

Overall the data indicated that a much higher concentration of17-β-estradiol was delivered by the phase IIb aqueous formulation in theearly stages of delivery. The middle stages of delivery (30 to 180minutes) show a “maintenance” level that is significantly different whencomparing the aqueous formulation (0.08%) to the timed releaseformulations including Carbopol+P3 formulation (0.034%) and theXanthan+CD formulation (0.16%). This data is also represented in Table10 below. In general, the data indicated the following:

-   -   The aqueous formulation and the Xanthan P3 formulation had        similar erosion profiles.    -   The timed release Carbopol CD formulation showed an unexpected        profile as it increased over time. This may be an artifact of        the combined release profile of the drug diffusing out of the CD        into the gel matrix and out of the matrix into the simulated        tear film.    -   The timed release Xanthan CD formulation had a higher        concentration than the aqueous formulation. This advantage        disappeared after 3 hours which is probably an indication that        the xanthan had eroded away and no longer provided a reservoir        for the drug.    -   The timed release Carbopol P3 formulation appeared to give the        best results with a huge difference in concentration even after        6 hours.

TABLE 10 Eroded Solution concentration (mg/mL) 2B Time (mins) (STD) CP3C-CD X CD X P3 15 0.01652 0.00095 0.00026 0.00080 0.00258 30 0.000260.00036 0.00015 0.00016 0.00009 45 0.00006 0.00014 0.00007 0.000090.00004 60 0.00002 0.00016 0.00006 0.00010 0.00005 75 0.00002 0.000070.00005 0.00013 0.00004 90 0.00002 0.00011 0.00003 0.00008 0.00003 1050.00003 0.00001 0.00003 0.00003 120 0.00002 0.00022 0.00030 0.000020.00002 150 0.00003 0.00010 0.00038 0.00003 0.00004 180 0.00004 0.000080.00007 0.00004 0.00005 210 0.00005 0.00010 0.00004 240 270 0.000070.00006 0.00005 300 330 360 0.00008 0.00009 0.00007

Results—Objective #2

The second objective was to achieve an equivalent or improved amount ofcross-corneal penetration when compared to the aqueous (Phase IIb—0.1%17-β-estradiol) formulation which has been proven efficacy. FIG. 12compares the concentration at different time points for the aqueouscontrol formulation and novel formulations. Initial runs were executedfor a period of 180 minutes. In all cases, the improved formulationshowed some degree of improved cross-corneal penetration when comparedto the aqueous Phase IIb formulation. Notably this improvement inconcentration crossing the cornea occurred even though the novel timedrelease formulations contained only twenty to thirty percent of theconcentration of active ingredient found in aqueous Phase IIbformulation.

The preliminary runs were executed through 180 minutes. Due to theunexpected notable improvements, the third study was executed through360 minutes. FIG. 13 shows the plot of the formulations that were runfor 6 hours. (Note: The data plotted in FIGS. 12 and 14 include theaveraged data for all points that had more than one data pointavailable).

FIG. 13 compares the concentration at different time points for thecontrol aqueous formulations and the main novel timed releaseformulations. The control aqueous formulation shows a rapid increasefollowed by a slow decline. The timed release formulations containingcarbopol-P3 and Xanthan with cyclodextrin were very similar in theearlier phase (first 2 hours) showing a slower build up to aconcentration that was similar to the peak concentration of the controlaqueous formulation. The timed release carbopol-P3 formulation thenstabilized and the concentration was maintained for the duration of theobservation period. Predictably, the concentration for the controlaqueous formulation declined after its peak. The performance for thetimed release formulation of Xanthan with cyclodextrin was quiteremarkable: it was still demonstrating increasing concentrations for thewhole observation period.

In terms of Area Under the Curve (AUC), if the control aqueousformulation is taken as standard as allocated a value of 1 for AUC, thenthe value for the timed release carbopol-P3 formulation was 1.21, whilethat for the timed release Xanthan gum with cyclodextrin formulation was2.18.

If the concentrations of the formulations were identical, then theresult would be similar to those reported for timolol. A differentpicture develops when one takes the concentration of the testformulations into account. The concentration of the timed releasecarbopol-P3 formulation was 0.033% 17-β-estradiol making the relativebioavailability in this model 3.6 times that for the control aqueousformulation. The timed release Xanthan gum with cyclodextrin formulationwith its concentration of 0.016% 17-β-estradiol has a relativebioavailability of 13.63 compared to the standard formulation! If theseresults are reproduced in the clinical setting as expected, the timedrelease Xanthan with cyclodextrin formulation would have a relativelybioavailability that is an amazing 13-14 times that for the standardformulation!

Results—Objective 3

The third objective was to reduce the peak amount of 17-β-estradiolbeing eliminated through the nasolacrimal drainage system, which can beshown by a slower rate of erosion in the Franz Cell model.

As discussed under objective 1 above, the aqueous formulation from thePhase IIb trial loses on average 74% of the applied dose (greater than80%, in one run) within the first fifteen minutes of application. FIG.14 presents a comparison of erosion data for all formulations tested.This analysis plots the cumulative percent of drug that is found exitingthe diffusion erosion chamber. This data models the concentrations ofdrug one expects to be eliminated through both reflux and normal tearflow.

The data confirmed the rapid erosion of almost 90% of the aqueouscontrol formulation compared with the timed release formulations beingtested. In clinical trials, the aqueous control formulation led to apeak systemic concentration of 102 pg/ml in post-menopausal women withdry eye, an increase of approximately 97 pg/ml over baseline values.Extrapolating from the Franz cell model, the increase in peak bloodestradiol concentration would be approximately 6 pg/ml with the timedrelease Xanthan with cyclodextrin formulation and approximately 3.6pg/ml for the timed release carbopol with cyclodextrin formulation. Thenormal in post-menopausal women is approximately 13 pg/ml. In theclinical trials the baseline value for estradiol in post-menopausalwomen with dry eyes was approximately 5 pg/ml. Thus, using either ofthese novel timed release formulations would not raise blood estradiollevels above the normal value.

CONCLUSION

In this Example 7, a number of novel timed release formulations weretested and compared with the standard aqueous formulation used in theclinical studies discussed in Example 2 above. The novel formulationscontaining cyclodextrin with Xanthan or carbopol achieved all thedesired goals.

The residence time on the ocular surface, represented in this model bythe concentration of estradiol in the erosion solution, showed the timedrelease Xanthan with cyclodextrin formulation to be superior to thestandard aqueous formulation over at least 3 hours. The timed releasecarbopol formulations were even better than this.

With respect to corneal penetration, the timed release carbopol withcyclodextrin formulation achieved a similar concentration as thestandard aqueous formulation. Unlike for the control aqueousformulation, these concentrations were maintained for the duration ofthe studies with no evidence of any decline. This was not expected. Thetimed release formulation of Xanthan gum with cyclodextrin achievedoutstanding corneal penetration concentrations which surprisingly werestill increasing six hours after instillation! Taking into account theconcentration of the formulations used, and using the AUC for theseproducts, the relative penetration is >3× and 13× that for the standardfor the timed release carbopol with cyclodextrin and the Xanthan gumwith cyclodextrin formulations, respectively.

The data confirmed that for the aqueous formulation approximately 90% ofthe delivered dose is eroded with most of the erosion occurring withinthe first 15 minutes. The timed release Xanthan gum with cyclodextrinformulation showed a cumulative erosion that never exceeded 35% withmost of this occurring in the first 2 hours. The erosion data for thetimed release carbopol with cyclodextrin formulation is even moreimpressive at approximately 10%, mostly within the first 2 hours. Thesevalues indicated that in clinical use, any increases in estradiol levelswill be minimal and should not exceed normal values.

The lowest concentration was that tested for the timed release Xanthangum with cyclodextrin formulation which had 0.016% 17-β-estradiol. Giventhe high corneal penetration achieved by this timed release formulation,it is likely that significantly lower concentrations than this willachieve similar efficacy as that reported for 0.1% 17-β-estradiolaqueous formulation. With concentrations as low as 0.005% 17-β-estradiolin the optimal timed release formulations, one should achieve similarcorneal penetration as the aqueous formulation at the efficacious 0.1%17-β-estradiol concentration.

What is claimed is:
 1. A topical application pharmaceutical composition for timed release comprising an estrogen receptor modulator at a concentration of less than 0.05% and a bioadhesive viscoelastic compound at a concentration sufficient to achieve timed release sufficient for delivery of a therapeutically effective concentration for alleviation of kerato-conjunctivitis sicca (dry eye syndrome) when administered twice daily or less frequently; wherein the bioadhesive viscoelastic compound comprises cyclodextrin and a compound selected from the group consisting of xanthan gum and a carbopol; and wherein the estrogen receptor modulator is 17-beta-estradiol or a derivative thereof.
 2. The topical application pharmaceutical of claim 1 wherein the pharmaceutical is in the form of a gel or a semi-solid ointment.
 3. The topical application pharmaceutical of claim 1 wherein the bioadhesive viscoelastic compound comprises xanthan gum.
 4. The topical application pharmaceutical of claim 1 wherein xanthan gum is present in an amount of between about 0.1% to about 1% (w/w).
 5. The topical application pharmaceutical of claim 1 wherein xanthan gum is present in an amount of about 0.6% (w/w).
 6. The topical application pharmaceutical of claim 5 wherein the bioadhesive viscoelastic compound comprises carbopol.
 7. The topical application pharmaceutical of claim 6 wherein the carbopol is carbopol 980 or carbopol
 940. 8. The topical application pharmaceutical of claim 6 wherein the carbopol is present in an amount of from about 0.5% to about 4% or about 2%.
 9. The topical application pharmaceutical of claim 1 wherein the cyclodextrin is present in an amount of from about 0.1% to about 7% (w/w).
 10. The topical application pharmaceutical of claim 1 wherein the cyclodextrin is present in an amount of from about 0.1% to about 1% (w/w).
 11. The topical application pharmaceutical of claim 1 wherein the cyclodextrin is present in an amount of about 0.4% (w/w).
 12. The topical application pharmaceutical of claim 1 wherein the timed release is sufficient for delivery of a therapeutically effective concentration for alleviation of kerato-conjunctivitis sicca (dry eye syndrome) when administered once per day or less frequently.
 13. The topical application pharmaceutical of claim 12 wherein the timed release is sufficient for delivery of a therapeutically effective concentration for alleviation of kerato-conjunctivitis sicca (dry eye syndrome) when administered once per day.
 14. The topical application pharmaceutical of claim 1 wherein the timed release comprises a controlled, sustained drug release rate which does not vary by more than about 50% for a period of at least about 8-12 hours after application.
 15. The topical application pharmaceutical of claim 1 wherein the timed release comprises a controlled, sustained drug release rate which delivers as measured by a Franz diffusion cell model with simulated tear flow a dose of between 0.1 mg/ml and 1.0 mg/ml of the estrogen receptor modulator per hour at least about 8-12 hours after application.
 16. The topical application pharmaceutical of claim 1 wherein the timed release comprises a controlled, sustained drug release rate which delivers a dose of between 0.1 pg and 1.0 pg of the estrogen receptor modulator per hour at least about 8-12 hours after application.
 17. The topical application pharmaceutical of claim 1 wherein the therapeutically effective amount is at least about 0.005% and less than 0.05% weight-by-weight of the estrogen receptor modulator.
 18. The topical application pharmaceutical of claim 1 wherein the increase in peak blood of the estrogen receptor modulator concentration after administration is less than 20 pg/ml.
 19. The topical application pharmaceutical of claim 1 wherein the average blood concentration of the estrogen receptor modulator after administration is less than 8 m I.U.
 20. A method of treatment comprising topical application to an eye of a topical application pharmaceutical of claim 1 for alleviation of kerato-conjunctivitis sicca (dry eye syndrome).
 21. The method of claim 20 wherein the kerato-conjunctivitis sicca is associated with post-menopausal subjects, premature ovarian failure, post-operative refractive surgery patients, corneal transplant patients or patients with other conditions that cause dry eye symptoms.
 22. The method of claim 20 wherein the topical application pharmaceutical is administered once per day or less frequently.
 23. The method of claim 22 wherein the topical application pharmaceutical is administered once per day.
 24. The method of claim 20 wherein the timed release comprises a controlled, sustained drug release rate which does not vary by more than about 50% for a period of at least about 8-12 hours after application.
 25. The method of claim 20 wherein the timed release comprises a controlled, sustained drug release rate which delivers as measured by a Franz diffusion cell model with simulated tear flow a dose of between 0.1 mg/ml and 1.0 mg/ml of the estrogen receptor modulator per hour at least about 8-12 hours after application.
 26. The method of claim 20 wherein the timed release comprises a controlled, sustained drug release rate which delivers a dose of between 0.1 pg and 1.0 pg of the estrogen receptor modulator per hour at least about 8-12 hours after application.
 27. The method of claim 20 wherein the therapeutically effective amount is at least about 0.005% and less than 0.05% weight-by-weight of the estrogen receptor modulator.
 28. The method of claim 20 wherein the increase in peak blood estradiol concentration after administration is less than 20 pg/ml.
 29. The method of claim 20 wherein the average blood concentration of estradiol after administration is less than 8 m I.U. 